Method for detecting endometriosis in a patient sample

ABSTRACT

This invention provides a polynucleotide encoding Repro-EN-1.0 and IB1, polypeptides associated with endometriosis. Auto-antibodies against Repro-EN-1.0 and IB1 have been found in subjects diagnosed with endometriosis. This invention also provides methods of using this polynucleotide and polypeptide.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to co-pending application of Schneider etal., “Diagnosis Of Autoimmune Disease By Detecting IgE or IgG₄,Autoantibodies Against Autoantigens, ”filed on even date herewith, thecontent of which is incorporated herein by reference in its entirety.

This application is a continuation of U.S. patent application Ser. No.11/168,187 filed Jun. 27 2005 now abandoned which is a divisional ofU.S. patent application Ser. No. 10/172,573 filed Jun. 13, 2002, andissued as U.S. Pat. No. 7,368,533, which, in turn, is a divisional ofU.S. patent application Ser. No. 09/447,399, filed Nov. 23, 1999, andissued as U.S. Pat. No. 6,525,187, which in turn is a continuation inpart of U.S. patent application Ser. No. 09/359,084 filed Jul. 22, 1999,now abandoned which, in turn, is a continuation of Provisional PatentApplication Ser. No. 60/094,930, filed Jul. 31, 1998, all of which areincorporated herein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Certain work described herein was supported by SBIR grant no. 1R43 HD33022-01A2 between the United States Department of Health and HumanServices and Reprogen, Inc. The Government may have certain rights inthis invention.

FIELD OF THE INVENTION

This invention is directed to the field of molecular biology in general,and, more specifically, to a polypeptide associated with endometriosis,an isolated polynucleotide encoding the polypeptide, and methods ofusing these molecules.

BACKGROUND OF THE INVENTION

Endometriosis is a painful disorder that is characterized by the ectopicimplantation of functioning endometrial tissue into the abdominal walland the outer surface of various organs including, most commonly, thelower bowel, ovaries and fallopian tubes. P. Vigano et al. (1991)Fertility and Sterility 56:894. Currently, endometriosis-specific geneshave not been identified and the events relating to the development ofendometriosis are poorly understood. However, several reports suggestthat retrograde menstruation linked with abnormal immune function mayplay a role in establishing ectopic endometrium lesions. T. Ishimaru andH. Masuzaki (1991) Am. J. Obstet. Gynecol. 165:210-214. Many attempts toisolate antigens from ectopic endometrium lesions have failed, due tothe necrotic nature of the lesions.

Endometriosis also is recognized has having an autoimmune component. IgGand IgA auto-antibodies that react with multiple endometrial antigenshave been documented in patients with endometriosis. However, attemptsto develop IgG or IgA-based assays for the diagnosis of endometriosishas fallen short of fruition. S. Fernandez-Shaw et al., (1996) Hum.Reprod. 11:180-1184. R. A. Wild et al. (1991) Obstetrics and Gynecology77:927. Studies have shown that circulating IgG antibodies that bindmultiple endometrial proteins can be detected in women withendometriosis to varying degrees. Thirty-five percent to 74% of patientshave sera reactive with endometrial proteins. O. Odukoya et al. (1996)Acta Obstet. Gynecol. Scand. 75:927-931; J. G. Kim et al. (1995) Am. J.Reprod. Immunol. 34:80-87; O. A. Odukoya et al. (1995) Hum. Reprod.10:1214-1219. It has also been shown that endometrial antibody titers inpatients that respond well to danazol are significantly lower ( 7/18(39%) treated patients had elevated titers) than those patients withuntreated endometriosis or patients that responded poorly to treatment (17/23 (74%) untreated patients had elevated titers). A. El-Roeiy et al.(1988) Fertility and Sterility 50:864-871; H. J. Chihal et al. (1986)Fertility and Sterility 46:408-411. In addition, it has been recentlyreported that women with endometriosis have elevated levels of IL-4, aTh2 mediating cytokine, and that treatment with danazol reduces thelevels of IL-4 in women that respond well to treatment. C.-C. Hsu et al.(1997) Fertility and Sterility 67:1059-1064.

SUMMARY OF THE INVENTION

This invention provides an isolated cDNA molecule and an alternatelyspliced variant encoding autoantigens associated with endometriosis. Theautoantigen is called Repro-EN-1.0. The alternately spliced variant iscalled IB1. Subjects diagnosed with endometriosis have been found tohave antibodies that specifically bind to Repro-EN-1.0 polypeptideand/or a IB1 polypeptide. These antibodies represent a highly sensitiveand specific diagnostic marker for endometriosis. RecombinantRepro-EN-1.0 protein and recombinant IB1 protein are useful to detectsuch antibodies in immunoassays.

In one aspect this invention provides a recombinant polynucleotidecomprising a nucleotide sequence encoding a polypeptide epitope of atleast 5 amino acids of Repro-EN-1.0 (SEQ ID NO:2), or IB1 (SEQ ID NO:4)wherein the epitope specifically binds to antibodies from subjectsdiagnosed with endometriosis. In one embodiment the nucleotide sequenceis selected from the Repro-EN-1.0 sequence of SEQ ID NO:1 or IB1sequence of SEQ ID NO: 3. In another embodiment the nucleotide sequenceis identical to nucleotides 176 to 2755 of SEQ ID NO:1 or nucleotides176 to 2986 of SEQ ID NO:3. In another embodiment the polynucleotidefurther comprises an expression control sequence operatively linked tothe nucleotide sequence.

In another aspect this invention provides a polynucleotide primer pairwhich amplifies a nucleotide sequence encoding a polypeptide epitope ofat least 5 amino acids of Repro-EN-1.0, or IB1 wherein the epitopespecifically binds to antibodies from subjects diagnosed withendometriosis. The pair comprises: 1) a 3′ primer of at least 7nucleotides that specifically hybridizes to a 3′ end of the nucleotidesequence or downstream from the sequence, and 2) a 5′ primer of at least7 nucleotides that specifically hybridizes to the 3′ end of thecomplement of the nucleotide sequence or downstream from the complementof the sequence.

In another aspect this invention provides a recombinant cell comprisinga recombinant polynucleotide comprising an expression control sequenceoperatively linked to a nucleotide sequence encoding a polypeptideepitope of at least 5 amino acids of Repro-EN-1.0 (SEQ ID NO:2), or IB1(SEQ ID NO:4), wherein the epitope specifically binds to antibodies fromsubjects diagnosed with endometriosis.

In another aspect this invention provides a method for detecting atarget polynucleotide comprising a nucleotide sequence selected fromRepro-EN-1.0 cDNA (SEQ ID NO: 1) or its complement, or IB1 cDNA (SEQ IDNO:3) or its complement in a sample. The method comprises the steps of:(a) contacting the sample with a polynucleotide probe or primercomprising a sequence of at least 7 nucleotides that specificallyhybridizes to the nucleotide sequence and (b) detecting whether theprobe or primer has specifically hybridized to the targetpolynucleotide, whereby specific hybridization provides a detection ofthe target polynucleotide in the sample.

In another aspect this invention provides a purified, recombinantRepro-EN-1.0 polypeptide whose amino acid sequence is identical to thatof SEQ ID NO:2, or an allelic variant of SEQ ID NO:2, or an IB1polypeptide whose amino acid sequence is identical to that of SEQ IDNo:4, or an allelic variant of SEQ ID: No:4.

In another aspect this invention provides a purified polypeptidecomprising an epitope of at least 5 amino acids of Repro-EN-1.0 (SEQ IDNO:2), or an epitope of at least 5 amino acids of IB1 (SEQ ID NO:4),wherein the epitope specifically binds to antibodies from subjectsdiagnosed with endometriosis.

In another aspect this invention provides a composition consistingessentially of an antibody that specifically binds to Repro-EN-1.0polypeptide (SEQ ID NO: 2), or IB1 polypeptide (SEQ ID NO:4).

In another aspect this invention provides a method for detecting aRepro-EN-1.0 polypeptide or IB1 polypeptide in a sample. The methodcomprises the steps of: (a) contacting the sample with a ligand thatspecifically binds to the Repro-EN-1.0 polypeptide or IB1 polypeptideand (b) detecting specific binding between the ligand and Repro-EN-1.0polypeptide or IB1 polypeptide. Specific binding provides a detection ofRepro-EN-1.0 polypeptide or IB1 polypeptide in the sample. In oneembodiment, the ligand is an antibody.

In another aspect this invention provides a method diagnosingendometriosis in a subject. The method comprises the steps of: (a)detecting a test amount of an antibody that specifically binds toRepro-EN-1.0 polypeptide or IB1 polypeptide in a sample from thesubject; and (b) comparing the test amount with a normal range of theantibody in a control sample from a subject who does not suffer fromendometriosis. A test amount above the normal range provides a positiveindication in the diagnosis of endometriosis. The sample can be a bloodproduct, e.g., serum, peritoneal fluid, menstrual fluid, vaginalsecretion or urine. In one embodiment the antibody is an IgE, IgG orIgG₄ immunoglobulin. In another embodiment the step of detectingcomprises capturing the antibody from the sample with an immobilizedRepro-EN-1.0 or a peptide comprising an epitope of Repro-EN-1.0 or IB1or a peptide comprising an epitope of IB1, and detecting capturedantibody. The step of detecting captured antibody can comprisecontacting the captured antibody with a detectable antibody thatspecifically binds immunoglobulins and detecting binding between thecaptured antibody and the detectable antibody. In another embodiment thestep of detecting can comprise capturing the antibody from the samplewith an immobilized anti-immunoglobulin antibody and detecting capturedantibody. The step of detecting captured antibody can comprisecontacting the captured antibody with Repro-EN-1.0 or a polypeptide, orIB1 or a peptide comprising an epitope of IB1, comprising an epitope ofRepro-EN-1.0 and detecting binding between the captured antibody and theRepro-EN-1.0 or polypeptide or IB1 or polypeptide.

In another aspect this invention provides a method for use in followingthe progress of endometriosis in a subject. The method comprises thesteps of: (a) detecting first and second amounts of an antibody thatspecifically bind Repro-EN-1.0 polypeptide, or IB1 polypeptide, insamples from the subject at a first and a second time, respectively; and(b) comparing the first and second amounts. An increase between thefirst and second amounts indicates progression of the endometriosis anda decrease between the first and second amounts indicates remission ofthe endometriosis.

In another aspect this invention provides an isolated MHC-peptidecomplex comprising at least a portion of an MHC Class I molecule or anMHC Class II molecule, wherein the portion comprises a binding site thatspecifically binds a peptide having an amino acid binding motif specificto the molecule, and wherein the portion engages in CD4-mediated orCD8-mediated binding to T cells, and a peptide of at least 8 amino acidsin a sequence selected from the amino acid sequence of Repro-EN-1.0 (SEQID NO:2) or IB1 (SEQ ID NO:4), wherein the peptide comprises the aminoacid binding motif and comprises an epitope that specifically binds to aT cell receptor; wherein the complex specifically binds a T cell havinga T cell receptor that specifically binds to the epitope, and whereinspecific binding induces anergy in the T cell.

In another aspect this invention provides a method for treatingendometriosis in a subject comprising the step of inhibiting an immuneresponse against Repro-EN-1.0 or IB1 in the subject. The method cancomprise administering to the subject an immunosuppressant in an amounteffective to inhibit the immune response, administering to the subjectan isolated MHC-peptide complex of the invention in an amount effectiveto inhibit the immune response or administering to the subject ananti-idiotypic antibody that specifically binds to an antigen bindingsite of an antibody that specifically binds to Repro-EN-1.0 or IB1 in anamount effective to inhibit the immune response.

In another aspect this invention provides a screening method fordetermining whether a compound increases or decreases the expression ofRepro-EN-1.0 in a cell comprising contacting the cell with the compoundand determining whether the production of Repro-EN-1.0 mRNA orpolypeptide, or IB1 mRNA or polypeptide, are increased or decreased.

In another aspect this invention provides a method of detecting achromosomal translocation of a Repro-EN-1.0 gene or IB1 gene comprisingthe steps of: a) hybridizing a labeled polynucleotide probe thatspecifically hybridizes with the Repro-EN-1.0 nucleotide sequence of SEQID NO:1 or its complement, or IB1 nucleotide sequence of SEQ ID NO:3 orits compliment, to a chromosome spread from a cell sample to determinethe pattern of hybridization and b) determining whether the pattern ofhybridization differs from a normal pattern. A difference in the patternprovides detection of a translocation.

In another aspect this invention provides a method of detectingpolymorphic forms of Repro-EN-1.0 or IB1 comprising the steps of: a)determining the identity of a nucleotide or amino acid at a selectedposition within the sequence of a test Repro-EN-1.0 gene or polypeptide,IB1 gene or polypeptide; b) determining the identity of the nucleotideor amino acid at the corresponding position of native Repro-EN-1.0 (SEQID NO: 1 or 2) gene or polypeptide, or IB1 (SEQ No:3 or 4) gene orpolypeptide; and c) comparing the identity from the test gene orpolynucleotide with the identity of the native gene or polypeptide,whereby a difference in identity indicates that the test polynucleotideis a polymorphic form of Repro-EN-1.0 or IB1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a northern blot analysis of Repro-EN-1.0 expression in varioustissues.

FIG. 2 is a northern blot analysis of Repro-EN-1.0 expression comparingvarious normal v. cancerous tissues.

FIG. 3 is a northern blot analysis of Repro-EN-1.0 expression in varioustissue culture cells.

DETAILED DESCRIPTION OF THE INVENTION I Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

“Polynucleotide” refers to a polymer composed of nucleotide units.Polynucleotides include naturally occurring nucleic acids, such asdeoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well asnucleic acid analogs. Nucleic acid analogs include those which includenon-naturally occurring bases, nucleotides that engage in linkages withother nucleotides other than the naturally occurring phosphodiester bondor which include bases attached through linkages other thanphosphodiester bonds. Thus, nucleotide analogs include, for example andwithout limitation, phosphorothioates, phosphorodithioates,phosphorotriesters, phosphoramidates, boranophosphates,methylphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand having the same sequence as an mRNAtranscribed from that DNA and which are located 5′ to the 5′-end of theRNA transcript are referred to as “upstream sequences”; sequences on theDNA strand having the same sequence as the RNA and which are 3′ to the3′ end of the coding RNA transcript are referred to as “downstreamsequences.”

“Complementary” refers to the topological compatibility or matchingtogether of interacting surfaces of two polynucleotides. Thus, the twomolecules can be described as complementary, and furthermore, thecon-tact surface characteristics are complementary to each other. Afirst polynucleotide is complementary to a second polynucleotide if thenucleotide sequence of the first polynucleotide is identical to thenucleotide sequence of the polynucleotide binding partner of the secondpolynucleotide. Thus, the polynucleotide whose sequence 5′-TATAC-3′ iscomplementary to a polynucleotide whose sequence is 5′-GTATA-3′.

A nucleotide sequence is “substantially complementary” to a referencenucleotide sequence if the sequence complementary to the subjectnucleotide sequence is substantially identical to the referencenucleotide sequence.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell. A host cell thatcomprises the recombinant polynucleotide is referred to as a“recombinant host cell.” The gene is then expressed in the recombinanthost cell to produce, e.g., a “recombinant polypeptide. ” A recombinantpolynucleotide may serve a non-coding function (e.g., promoter, originof replication, ribosome-binding site, etc.) as well.

“Expression control sequence” refers to a nucleotide sequence in apolynucleotide that regulates the expression (transcription and/ortranslation) of a nucleotide sequence operatively linked thereto.“Operatively linked” refers to a functional relationship between twoparts in which the activity of one part (e.g., the ability to regulatetranscription) results in an action on the other part (e.g.,transcription of the sequence). Expression control sequences caninclude, for example and without limitation, sequences of promoters(e.g., inducible or constitutive), enhancers, transcription terminators,a start codon (i.e., ATG), splicing signals for introns, and stopcodons.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and virusesthat incorporate the recombinant polynucleotide.

“Amplification” refers to any means by which a polynucleotide sequenceis copied and thus expanded into a larger number of polynucleotidemolecules, e.g., by reverse transcription, polymerase chain reaction,and ligase chain reaction.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

“Probe,” when used in reference to a polynucleotide, refers to apolynucleotide that is capable of specifically hybridizing to adesignated sequence of another polynucleotide. A probe specificallyhybridizes to a target complementary polynucleotide, but need notreflect the exact complementary sequence of the template. In such acase, specific hybridization of the probe to the target depends on thestringency of the hybridization conditions. Probes can be labeled with,e.g., chromogenic, radioactive, or fluorescent moieties and used asdetectable moieties.

A first sequence is an “antisense sequence” with respect to a secondsequence if a polynucleotide whose sequence is the first sequencespecifically hybridizes with a polynucleotide whose sequence is thesecond sequence.

“Hybridizing specifically to” or “specific hybridization” or“selectively hybridize to”, refers to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology Hybridization with Nucleic AcidProbes part I chapter 2 “Overview of principles of hybridization and thestrategy of nucleic acid probe assays”, Elsevier, N.Y. Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the thermal melting point (Tm) for the specific sequenceat a defined ionic strength and pH. The Tm is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Very stringent conditions areselected to be equal to the Tm for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al. for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. In general, a signal to noise ratio of 2× (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.The term “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

“Conservative substitution” refers to the substitution in a polypeptideof an amino acid with a functionally similar amino acid. The followingsix groups each contain amino acids that are conservative substitutionsfor one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

“Allelic variant” refers to any of two or more polymorphic forms of agene occupying the same genetic locus. Allelic variations arisenaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequences. “Allelic variants” also refer to cDNAs derived from mRNAtranscripts of genetic allelic variants, as well as the proteins encodedby them.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

The terms “identical” or percent “identity,” in the context of two ormore polynucleotide or polypeptide sequences, refer to two or moresequences or sub-sequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or sub-sequencesthat have at least 60%, 80%, 90%, 95% or 98% nucleotide or amino acidresidue identity, when compared and aligned for maximum correspondence,as measured using one of the following sequence comparison algorithms orby visual inspection. Preferably, the substantial identity exists over aregion of the sequences that is at least about 50 residues in length,more preferably over a region of at least about 100 residues, and mostpreferably the sequences are substantially identical over at least about150 residues. In a most preferred embodiment, the sequences aresubstantially identical over the entire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word-length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word-length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Proc. Natl. Acad.Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described herein.

An “affinity agent” is a compound that specifically or non-specificallybinds to a target molecule. Affinity agents that non-specifically bindto a molecule include, for example, anion or cation exchange resins, ormaterials that bind hydrophobic or hydrophilic molecules, or metal ions.

A “ligand” is a compound that specifically binds to a target molecule.

A “receptor” is compound that specifically binds to a ligand.

“Antibody” refers to a polypeptide ligand substantially encoded by animmunoglobulin gene or inimunoglobulin genes, or fragments thereof,which specifically binds and recognizes an epitope (e.g., an antigen).The recognized immunoglobulin genes include the kappa and lambda lightchain constant region genes, the alpha, gamma, delta, epsilon and muheavy chain constant region genes, and the myriad immunoglobulinvariable region genes. Antibodies exist, e.g., as intact immunoglobulinsor as a number of well characterized fragments produced by digestionwith various peptidases. This includes, e.g., Fab′ and F(ab)′2fragments. The term “antibody,” as used herein, also includes antibodyfragments either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA methodologies. It alsoincludes polyclonal antibodies, monoclonal antibodies, chimericantibodies and humanized antibodies. “Fc” portion of an antibody refersto that portion of an immunoglobulin heavy chain that comprises one ormore heavy chain constant region domains, CH, CH2 and CH3, but does notinclude the heavy chain variable region.

A ligand or a receptor (e.g., an antibody) “specifically binds to” or“is specifically immunoreactive with” a compound analyte when the ligandor receptor functions in a binding reaction which is determinative ofthe presence of the analyte in a sample of heterogeneous compounds.Thus, under designated assay (e.g., immunoassay) conditions, the ligandor receptor binds preferentially to a particular analyte and does notbind in a significant amount to other compounds present in the sample.For example, a polynucleotide specifically binds under hybridizationconditions to an analyte polynucleotide comprising a complementarysequence; an antibody specifically binds under immunoassay conditions toan antigen analyte bearing an epitope against which the antibody wasraised; and an adsorbent specifically binds to an analyte under properelution conditions.

“Immunoassay” refers to a method of detecting an analyte in a sample inwhich specificity for the analyte is conferred by the specific bindingbetween an antibody and a ligand. This includes detecting an antibodyanalyte through specific binding between the antibody and a ligand. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York, for a description of immunoassay formatsand conditions that can be used to determine specific immunoreactivity.

“Vaccine” refers to an agent or composition containing an agenteffective to confer a therapeutic degree of immunity on an organismwhile causing only very low levels of morbidity or mortality. Methods ofmaking vaccines are, of course, useful in the study of the immune systemand in preventing and treating animal or human disease.

An “immunogenic amount” is an amount effective to elicit an immuneresponse in a subject.

“Substantially pure” or “isolated” means an object species is thepredominant species present (i.e., on a molar basis, more abundant thanany other individual macromolecular species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50% (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition means that about 80% to 90% or more of the macromolecularspecies present in the composition is the purified species of interest.The object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) if the composition consists essentially of a singlemacromolecular species. Solvent species, small molecules (<500 Daltons),stabilizers (e.g., BSA), and elemental ion species are not consideredmacromolecular species for purposes of this definition.

“Naturally-occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring.

“Detecting” refers to determining the presence, absence, or amount of ananalyte in a sample, and can include quantifying the amount of theanalyte in a sample or per cell in a sample.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, ³⁵S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavadin, dioxigenin, haptens and proteins for which antiseraor monoclonal antibodies are available, or nucleic acid molecules with asequence complementary to a target. The detectable moiety oftengenerates a measurable signal, such as a radioactive, chromogenic, orfluorescent signal, that can be used to quantitate the amount of bounddetectable moiety in a sample. The detectable moiety can be incorporatedin or attached to a primer or probe either covalently, or through ionic,van der Waals or hydrogen bonds, e.g., incorporation of radioactivenucleotides, or biotinylated nucleotides that are recognized bystreptavadin. The detectable moiety may be directly or indirectlydetectable. Indirect detection can involve the binding of a seconddirectly or indirectly detectable moiety to the detectable moiety. Forexample, the detectable moiety can be the ligand of a binding partner,such as biotin, which is a binding partner for streptavadin, or anucleotide sequence, which is the binding partner for a complementarysequence, to which it can specifically hybridize. The binding partnermay itself be directly detectable, for example, an antibody may beitself labeled with a fluorescent molecule. The binding partner also maybe indirectly detectable, for example, a nucleic acid having acomplementary nucleotide sequence can be a part of a branched DNAmolecule that is in turn detectable through hybridization with otherlabeled nucleic acid molecules. (See, e.g., P D. Fahrlander and A.Klausner, Bio/Technology (1988) 6:1165.) Quantitation of the signal isachieved by, e.g., scintillation counting, densitometry, or flowcytometry.

“Linker” refers to a molecule that joins two other molecules, eithercovalently, or through ionic, van der Waals or hydrogen bonds, e.g., anucleic acid molecule that hybridizes to one complementary sequence atthe 5′ end and to another complementary sequence at the 3′ end, thusjoining two non-complementary sequences.

“Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in a mammal. A pharmaceutical composition comprises apharmacologically effective amount of an active agent and apharmaceutically acceptable carrier. “Pharmacologically effectiveamount” refers to that amount of an agent effective to produce theintended pharmacological result. “Pharmaceutically acceptable carrier”refers to any of the standard pharmaceutical carriers, buffers, andexcipients, such as a phosphate buffered saline solution, 5% aqueoussolution of dextrose, and emulsions, such as an oil/water or water/oilemulsion, and various types of wetting agents and/or adjuvants. Suitablepharmaceutical carriers and formulations are described in Remington'sPharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995).Preferred pharmaceutical carriers depend upon the intended mode ofadministration of the active agent. Typical modes of administrationinclude enteral (e.g., oral) or parenteral (e.g., subcutaneous,intramuscular, intravenous or intraperitoneal injection; or topical,transdermal, or transmucosal administration). A “pharmaceuticallyacceptable salt” is a salt that can be formulated into a compound forpharmaceutical use including, e.g., metal salts (sodium, potassium,magnesium, calcium, etc.) and salts of ammonia or organic amines.

“Small organic molecule” refers to organic molecules of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes organic biopolymers (e.g., proteins, nucleic acids,etc.). Preferred small organic molecules range in size up to about 5000Da, up to about 2000 Da, or up to about 1000 Da.

A “subject” of diagnosis or treatment is a human or non-human mammal.Non-human mammals subject to diagnosis or treatment include, forexample, primates, ungulates, canines and felines.

“Treatment” refers to prophylactic treatment or therapeutic treatment.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

“Diagnostic” means identifying the presence or nature of a pathologiccondition. Diagnostic methods differ in their sensitivity andspecificity. The “sensitivity” of a diagnostic assay is the percentageof diseased individuals who test positive (percent of true positives).The “specificity” of a diagnostic assay is 1 minus the false positiverate, where the false positive rate is defined as the proportion ofthose without the disease who test positive. While a particulardiagnostic method may not provide a definitive diagnosis of a condition,it suffices if the method provides a positive indication that aids indiagnosis.

“Prognostic” means predicting the probable development (e.g., severity)of a pathologic condition.

“Test amount” refers to an amount of an analyte in a subject sample,which is then compared to a normal amount of the analyte in a sample(e.g., from a healthy individual) such that the relative comparison ofthe values provides a reference value for diagnosing a designateddisease. Depending upon the method of detection, the test amount may bea determination of the amount of the analyte, but it is not necessarilyan amount. The test amount may also be a relative value, such as a plusor a minus score, and also includes an amount indicating the presence orabsence of the analyte in a sample.

“Normal amount” refers to an amount or a range of an analyte in abiological sample that indicates health or lack of pathology.

“Diagnostic amount” refers to an amount of an analyte in a subjectsample that is consistent with a particular diagnosis for a designateddisease.

“Prognostic amount” refers to an amount or range of an analyte in asubject sample that is consistent with a particular prognosis for adesignated disease.

“Plurality” means at least two.

An “epitope” is portion of a molecule that specifically binds to anantibody or a T cell receptor. An peptide epitope generally comprises asequence of at least 6 amino acids from a polypeptide, although longerand shorter peptides can constitute epitopes.

“MHC Class I molecule” refers to a heterodimer found on the surface ofcells that present processed antigenic peptides to T cells. The moleculecomprises an α chain and a β-microglobulin chain. The α chain containsthe antigenic peptide binding site in the α1 and α2 domains. The α chainalso contains a transmembrane portion that can be removed withouteliminating antigen binding.

“MHC Class II molecule” refers to a heterodimer found on the surface ofcells that present processed antigenic peptide to T cells. It comprisesan α chain and a β chain. The antigenic peptide binding site is locatedin the α1 domain of the α chain and the β1 domain of the β chain.However, a single a chain or β chain suffices to bind an antigenicpeptide. The α chain and β chain also contain transmembrane regions thatcan be removed without eliminating antigenic peptide binding function.

“T cell receptor” refers to a heterodimer found on the surface of Tcells comprising an α chain and a β chain or a γ and a δ chain. T cellreceptors recognize processed antigens associated with MHC molecules.

II cDNA Encoding Repro-EN-1.0 and IB1

We have isolated a cDNA molecule encoding an autoantigen associated withendometriosis. The autoantigen is called Repro-EN-1.0. The presence ofantibodies that specifically bind to an epitope of the Repro-EN-1.0polypeptide is a highly sensitive and specific diagnostic marker forendometriosis.

Polynucleotides encoding full-length Repro-EN-1.0 are useful inrecombinant production Repro-EN-1.0 or immunogenic fragments of it.Fragments of polynucleotides encoding Repro-EN-1.0 are useful as probesto detect Repro-EN-1.0 mRNA from certain cell types suspected to becancerous. Fragments also are useful as primers for amplification ofsequences from Repro-EN-1.0.

The Repro-EN-1.0 polypeptide and immunogenic fragments of it are usefulas positive controls in diagnostic assays to detect antibodies thatspecifically bind to Repro-EN-1.0 from patient serum samples. Thepolypeptides also are useful as immunogens for eliciting production ofantibodies against epitopes of the protein.

The nucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence(SEQ ID NO:2) of Repro-EN-1.0 follow:

This 3164-base nucleotide sequence contains an open reading frame of2580 nucleotides encoding Repro-EN-1.0 from nucleotide 176 to nucleotide2755. The deduced amino acid sequence of Repro-EN-1.0 has 860 aminoacids. Repro-EN-1.0 has a calculated molecular mass of 96.4 kD and a pIof 5.08.

The Repro-EN-1.0 gene encodes a 3.4 kb mRNA. This mRNA is expressedprimarily in skeletal muscle, heart and testis, and to a lesser extentin other tissues. However, it is not detected in lung or peripheralblood mononuclear cells (PBMC). Expression of Repro-EN-1.0 isup-regulated in breast and uterine carcinomas relative to their normalcounterparts. It is highly expressed in both normal fallopian tube andfallopian tube carcinoma. It is expressed in low levels in normal ovaryand ovarian carcinoma. Expression of the mRNA is lower in endometrialcarcinoma cell lines than in prostate adenocarcinoma cell lines.

Analysis of the deduced amino acid sequence of Repro-EN-1.0 shows nosignificant sequence identity with any other protein.

There is an alternately spliced variant that was isolated form a humanheart cDNA library. This variant is called IB1 and is useful in the sameways as the nucleotide sequence (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of Repro-EN-1.0. The IB1 sequence was isolatedfrom a heart cDNA library by screening with a nucleic acid probeobtained from the Repro-EN-1.0 sequence. The IB1 sequence is differentfrom the Repro-EN-1.0 sequence in that it contains an additional 231 byexon inserted into the cDNA sequence at position 1555. Therefore the IB1sequence has similar properties, but is slightly larger.

The nucleotide sequence (SEQ ID NO:3) and the deduced amino acidsequence (SEQ ID NO:4) of IB1 follow:

IB1 Alternately Spliced Variant from Heart Library:

IB1 cDNA is 3,395 base pairs

IB1 open reading frame is 2,811 nucleotides

IB1 open reading frame maps to nucleotides 176 to 2986

Deduced amino acid sequence of IB1 has 937 amino acids

Calculated molecular mass of IB1 protein is 104,969 Daltons (105 kD)

Calculated pI of IB1 protein is 5.17

Analysis of the amino acid sequence identified several amino acid motifsthat will be apparent to those skilled in the art including a myb 1 DNAbinding domain, a WD 40 site, an RGD cell-attachment sequence, anN-myristolylation site and several phosphorylation and glycosylationsites. Analysis also shows that the protein is largely hydrophilic. Thisimplies that most of the amino acid sequence is exposed to the immunesystem and can be recognized as epitopes.

Analysis of expressed sequence tags (ESTs) from a public database(Genbank) identified many overlapping ESTs that, together, covered mostof the Repro-EN-1.0 cDNA sequence.

III. Repro-EN-1.0 and IB1 Nucleic Acids

This invention provides recombinant polynucleotides comprising anucleotide sequence encoding Repro-EN-1.0 and IB1 proteins, Repro-EN-1.0and IB1 analogs or fragments of these polypeptides, as described herein.Repro-EN-1.0 or IB1 analogs include 1) immunogenic fragments ofRepro-EN-1.0 or IB1; 2) homologs of Repro-EN-1.0 or IB1 from othermammals (especially primates); 3) fragments of Repro-EN-1.0 or IB1comprising at least 5 consecutive amino acids from the sequence ofRepro-EN-1.0 or IB1; 4) non-naturally occurring polypeptides whosesequences are substantially identical to Repro-EN-1.0; and 5) fusionproteins comprising a Repro-EN-1.0 or IB1 or a Repro-EN-1.0 or IB1analog fused to a second polypeptide moiety. The polynucleotides areuseful for expressing the mRNA or polypeptides they encode and in thepreparation of probes or primers, among other things.

In one embodiment, the recombinant polynucleotide molecule comprises anucleotide sequence encoding a sequence of at least 5 amino acidsselected from the amino acid sequence of Repro-EN-1.0 (SEQ ID NO:2) orIB1 (SEQ ID NO:4). The nucleotide sequence can encode a sequence of atleast 25 amino acids, at least 100 amino acids or at least 200 aminoacids from SEQ ID NO:2 or SEQ ID NO:4. In one embodiment, the nucleotidesequence encodes an immunogenic analog. One such immunogenic analog is apolypeptide comprising an epitope that binds specifically to an antibodyfrom serum from a subject diagnosed with endometriosis. In anotherembodiment, the nucleotide sequence encodes full-length nativeRepro-EN-1.0 or IB1 polypeptide.

The nucleotide sequence can be identical to a sequence from Repro-EN-1.0cDNA or its complement or IB1 cDNA or its complement, or can includedegenerate codons. In one embodiment of a nucleotide sequence encodingfull-length Repro-EN-1.0 or IB1, the sequence is identical to the codingsequence of Repro-EN-1.0 of SEQ ID NO: 1 or IB1 of (SEQ ID NO:3). Inanother embodiment, the nucleotide sequence encodes a Repro-EN-1.0 orIB1 analog whose amino acid sequence is substantially identical to theamino acid sequence of Repro-EN-1.0 polypeptide (SEQ ID NO: 2) or IB1polypeptide (SEQ ID NO:4).

In another embodiment, the polynucleotide encodes a fusion proteinbetween Repro-EN-1.0 or IB1 polypeptide or Repro-EN-1.0 or IB1 analogamino acid sequences and a second amino acid sequence. The second aminoacid sequence can be, for example, a detectable label such as afluorescent protein, enzyme marker of protein from a two-hybrid system.

The polynucleotides of the present invention are cloned or amplified byin vitro methods, such as the polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR) andthe Qβ replicase amplification system (QB). For example, apolynucleotide encoding the protein can be isolated by polymerase chainreaction of cDNA from a human endometrial carcinoma cell line usingprimers based on the DNA sequence of Repro-EN-1.0 of SEQ ID NO:1. Onepair of primers useful for amplifying Repro-EN-1.0 DNA, includingallelic variants, is:

Upstream sense: 5′-caggacacagatgacagtgat-3′ (SEQ ID NO: 5) Downstreamantisense: 5′-agagccttctgatctgtcac-3′. (SEQ ID NO: 6)

A wide variety of cloning and in vitro amplification methodologies arewell-known to persons of skill. PCR methods are described in, forexample, U.S. Pat. No. 4,683,195; Mullis et al. (1987) Cold SpringHarbor Symp. Quant. Biol. 51:263; and Erlich, ed., PCR Technology,(Stockton Press, NY, 1989). One useful format is real time PCR. See,e.g., Luch et al. (1997) J. Molec. Endocrinol. 18:77-85 and Arold etal., (1997) Proc. Nat'l Acad. Sci., USA 94:2438-43. Polynucleotides alsocan be isolated by screening genomic or cDNA libraries with probesselected from the sequences of SEQ ID NO:1 under stringent hybridizationconditions.

Mutant versions of the proteins can be made by site-specific mutagenesisof other polynucleotides encoding the proteins, or by random mutagenesiscaused by increasing the error rate of PCR of the originalpolynucleotide with 0.1 mM MnCl2 and unbalanced nucleotideconcentrations.

This invention also provides expression vectors, e.g., recombinantpolynucleotide molecules comprising expression control sequencesoperatively linked to a nucleotide sequence encoding the targetpolypeptide. Expression vectors can be adapted for function inprokaryotes or eukaryotes by inclusion of appropriate promoters,replication sequences, markers, etc. for transcription and translationof mRNA. The construction of expression vectors and the expression ofgenes in transfected cells involves the use of molecular cloningtechniques also well known in the art. Sambrook et al., MolecularCloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., (1989) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., (Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc.) Usefulpromoters for such purposes include a metallothionein promoter, aconstitutive adenovirus major late promoter, a dexamethasone-inducibleMMTV promoter, a SV40 promoter, a MRP polIII promoter, a constitutiveMPSV promoter, a tetracycline-inducible CMV promoter (such as the humanimmediate-early CMV promoter), and a constitutive CMV promoter.

Methods for transfecting genes into mammalian cells and obtaining theirexpression for in vitro use or for gene therapy, are well known to theart. See, e.g., Methods in Enzymology, vol. 185, Academic Press, Inc.,San Diego, Calif. (D. V. Goeddel, ed.) (1990) or M. Krieger, GeneTransfer and Expression—A Laboratory Manual, Stockton Press, New York,N.Y., (1990).

Expression vectors useful in this invention depend on their intendeduse. Such expression vectors must, of course, contain expression andreplication signals compatible with the host cell. Expression vectorsuseful for expressing the protein of this invention include viralvectors such as alpha viruses, retroviruses, adenoviruses andadeno-associated viruses, plasmid vectors, cosmids, liposomes and thelike. Viral and plasmid vectors are preferred for transfecting mammaliancells. The expression vector pcDNA1 (Invitrogen, San Diego, Calif.), inwhich the expression control sequence comprises the CMV promoter,provides good rates of transfection and expression. Adeno-associatedviral vectors are useful in the gene therapy methods of this invention.

The construct can also contain a tag to simplify isolation of theprotein. For example, a polyhistidine tag of, e.g., six histidineresidues, can be incorporated at the amino terminal end of the protein.The polyhistidine tag allows convenient isolation of the protein in asingle step by nickel-chelate chromatography.

In another embodiment, endogenous genes are transcribed by operativelylinking them to expression control sequences supplied endogenously thatrecombine with genomic DNA. In one method, one provides the cell with arecombinant polynucleotide containing a targeting sequence, whichpermits homologous recombination into the genome upstream of thetranscriptional start site of target gene; the expression controlsequences; an exon of the target gene; and an unpaired splice-donor sitewhich pairs with a splice acceptor in the target gene. Such methods arediscussed in Treco et al., WO 94/12650; Treco et al., WO 95/31560 andTreco et al., WO 96/29411.

The invention also provides recombinant cells comprising an expressionvector for expression of the nucleotide sequences encoding a polypeptideof this invention. Host cells can be selected for high levels ofexpression in order to purify the protein. Mammalian cells are preferredfor this purpose, but prokaryotic cells, such as E. coli, also areuseful. The cell can be, e.g., a recombinant cell in culture or a cellin vivo.

IV Polynucleotide Probes and Primers

This invention provides polynucleotide probes and primers thatspecifically hybridize to a sub-sequence of Repro-EN-1.0 cDNA or itscomplement or IB1 cDNA or its complement, under stringent hybridizationconditions. The probes and primers of this invention are polynucleotidesof at least 7 nucleotides, at least 10 nucleotides, at least 15nucleotides, at least 20 nucleotides or at least 25 nucleotides. In oneembodiment, the sequence of the polynucleotide is a contiguous sequencefrom SEQ ID NO:1 or its complement. Any suitable region of theRepro-EN-1.0 or IB1 gene may be chosen as a target for polynucleotidehybridization. Nucleotide substitutions, deletions, and additions may beincorporated into the polynucleotides as long as the characteristicability to specifically hybridize to the target sequence or itscomplement is retained. Nucleotide sequence variation may result fromsequence polymorphisms of various alleles, minor sequencing errors, andthe like.

The probes and primers of the invention are useful as probes inhybridization assays, such as Southern and northern blots, foridentifying polynucleotides having a nucleotide sequence encoding aRepro-EN-1.0 or IB1 polypeptide, and as primers for amplificationprocedures. The probes and primers of the invention are also useful indetecting the presence, absence or amount of Repro-EN-1.0 or IB1 intissue biopsies and histological sections where the detection method iscarried out in situ, typically after amplification of Repro-EN-1.0 orIB1 sequences using a primer set.

The probes and primers of this invention also are useful for identifyingallelic forms of Repro-EN-1.0 and animal cognate genes or IB1 and animalcognate genes. Probes and primers can be used to screen human or animalgenomic DNA or cDNA libraries under, e.g., stringent conditions. DNAmolecules that specifically hybridize to the probe are then furtherexamined to determine whether they are Repro-EN-1.0 allelic variants oranimal cognates or IB1 allelic variants or animal cognates.

The probes also are useful in oligonucleotide arrays. Such arrays areused in hybridization assays to check the identity of bases in a targetpolynucleotide. In essence, when a target hybridizes perfectly to aprobe on the array, the target contains the nucleotide sequence of theprobe. When the target hybridizes less well, or does not hybridize atall, then the target and probe differ in sequence by one or morenucleotide. By proper selection of probes, one can check bases on atarget molecule. See, e.g., Chee et al., WO 95/11995. The use theRepro-EN-1.0 or IB1 sequence in genomics is described further below.

In one embodiment, the polynucleotide is directly or indirectlydetectable through a detectable moiety. A detectable moiety bound toeither an oligonucleotide primer or a probe is subsequently used todetect hybridization of an oligonucleotide primer to the RNA component.Detection of labeled material bound to a Repro-EN-1.0 or IB1polynucleotide in a sample provides a means of determining a diagnosticor prognostic value.

Although primers and probes can differ in sequence and length, theprimary differentiating factor is one of function: primers serve as aninitiation point for DNA synthesis of a target polynucleotide, as in RTand PCR reactions, while probes are typically used for hybridization toand detection of a target polynucleotide. Typical lengths of primers orprobes can range from 7-50 nucleotides, preferably from 10-40nucleotides, and most preferably from 15-35 nucleotides. A primer orprobe can also be labeled with a detectable moiety for detection ofhybridization of the primer or probe to the target polynucleotide.

In general, those of skill in the art recognize that the polynucleotidesused in the invention include both DNA and RNA molecules and naturallyoccurring modifications thereof, as well as synthetic, non-naturallyoccurring analogs of the same, and heteropolymers, ofdeoxyribonucleotides, ribonucleotides, and/or analogs of either. Theparticular composition of a polynucleotide or polynucleotide analog willdepend upon the purpose for which the material will be used and theenvironment in which the material will be placed. Modified or synthetic,non-naturally occurring nucleotides have been designed to serve avariety of purposes and to remain stable in a variety of environments,such as those in which nucleases are present.

Oligonucleotides preferably are synthesized, e.g., on an AppliedBioSystems or other commercially available oligonucleotide synthesizeraccording to specifications provided by the manufacturer.Oligonucleotides may be prepared using any suitable method, such as thephosphotriester and phosphodiester methods, or automated embodimentsthereof. In one such automated embodiment, diethylphosphoramidates areused as starting materials and may be synthesized as described byBeaucage et al., Tetrahedron Letters 22: 1859 (1981), and U.S. Pat. No.4,458,066.

Polynucleotides, e.g., probes, also can be recombinantly producedthrough the use of plasmids or other vectors.

In one aspect this invention provides a probe that specificallyhybridizes to the 5′ untranslated region of Repro-EN-1.0 or IB1, thecoding region of Repro-EN-1.0 or IB1, or a region of Repro-EN-1.0 or IB1encoding an epitope of the Repro-EN-1.0 or IB1 polypeptide.

In another aspect, this invention provides a primer pair which amplifiesa nucleotide sequence encoding a polypeptide epitope of Repro-EN-1.0 orIB1 recognized by an antibody from an individual diagnosed withendometriosis. A primer pair that amplifies a particular nucleotidesequence (given in the 5′ to 3′ orientation) includes a 5′ primer and a3′ primer. The 3′ primer hybridizes to the 3′ end of the nucleotidesequence or downstream from it. The 5′ primer hybridizes to the 3′ endof the complement of the nucleotide sequence or downstream from it. Inthis way, the primers can amplify a polynucleotide that comprises thenucleotide sequence. One nucleotide sequence encoding a polypeptideepitope of Repro-EN-1.0 has been identified within the about 2.2 kb from3′ end of the coding sequence of Repro-EN-1.0 (SEQ ID NO: 1).

V. Methods for Detecting Repro-EN-1.0 and IB1 Polynucleotides

The probes and primers of this invention are useful, among other things,in detecting Repro-EN-1.0 or IB1 polynucleotides in a sample. A methodfor detecting the presence, absence or amount of a Repro-EN-1.0 or IB1polynucleotide in a sample involves two steps: (1) specificallyhybridizing a polynucleotide probe or primer to a Repro-EN-1.0 or IB1polynucleotide, and (2) detecting the specific hybridization.

For the first step of the method, the polynucleotide used for specifichybridization is chosen to hybridize to any suitable region ofRepro-EN-1.0. The polynucleotide can be a DNA or RNA molecule, as wellas a synthetic, non-naturally occurring analog of the same. Thepolynucleotides in this step are polynucleotide primers andpolynucleotide probes disclosed herein. This includes probes and primershaving sequences selected from the sequence of Repro-EN-1.0 (SEQ IDNO:1) or selected from the cyber-sequence of Repro-EN-1.0 (SEQ ID NO:5).

For the second step of the reaction, any suitable method for detectingspecific hybridization of a polynucleotide to Repro-EN-1.0 or IB1 may beused. Such methods include, e.g., amplification by extension of ahybridized primer using reverse transcriptase (RT); extension of ahybridized primer using RT-PCR or other methods of amplification; and insitu detection of a hybridized primer. In in situ hybridization, asample of tissue or cells is fixed onto a glass slide and permeablizedsufficiently for use with in situ hybridization techniques. Detectablemoieties used in these methods include, e.g., labeled polynucleotideprobes; direct incorporation of label in amplification or RT reactions,and labeled polynucleotide primers.

Often, cell extracts or tissue samples used in methods for determiningthe amount of a polynucleotide in a sample will contain variable amountsof cells or extraneous extracellular matrix materials. Thus, a methodfor determining the cell number in a sample is important for determiningthe relative amount per cell of a test polynucleotide such asRepro-EN-1.0 or IB1. A control for cell number and amplificationefficiency is useful for determining diagnostic values for a sample of apotential cancer, and a control is particularly useful for comparing theamount of test polynucleotide such as Repro-EN-1.0 or IB1 in sample to adiagnostic value for breast cancer, uterine cancer or fallopian tubecancer. A preferred embodiment of the control RNA is endogenouslyexpressed 28S rRNA. (See, e.g., Khan et al., Neurosci. Lett. 147:114-117 (1992) which used 28S rRNA as a control, by diluting reversetranscribed 28S rRNA and adding it to the amplification reaction.)

VI. Inhibitory Polynucleotides for Inhibiting Repro-EN-1.0 and IB1Expression

A. General

This invention also provides inhibitory polynucleotides directed againstRepro-EN-1.0 or IB1 polynucleotides that inhibit Repro-EN-1.0 or IB1expression and, therefore inhibit its activity in a cell. Inhibitorypolynucleotides can inhibit Repro-EN-1.0 or IB1 activity in a number ofways. According to one mechanism, the polynucleotide preventstranscription of the Repro-EN-1.0 or IB1 gene (for instance, by triplehelix formation). In another mechanism, the polynucleotide destabilizesthe Repro-EN-1.0 or IB1 and reduces its half-life. In another mechanism,the polynucleotide inhibits assembly of the RNA component into theRepro-EN-1.0 or IB1 by binding to Repro-EN-1.0 or IB1.

An inhibitory polynucleotide is a polynucleotide that is capable ofspecifically hybridizing with a target polynucleotide and thatinterferes with the transcription, processing, translation or otheractivity the target polynucleotide. Inhibitory polynucleotides generallyare single-stranded and have a sequence of at least 7, 8, 9, 10, or 11nucleotides capable of specifically hybridizing to the target sequence.RNA sequences generally require a sequence of at least 10 nucleotidesfor specific hybridization. Inhibitory polynucleotides include, withoutlimitation, antisense molecules, ribozymes, sense molecules andtriplex-forming molecules. In one embodiment, the inhibitorypolynucleotide is no more than about 50 nucleotides long.

While not wishing to be limited by theory, it is believed thatinhibitory polynucleotides inhibit the function of a target, in part, bybinding to the appropriate target sequence. An inhibitory polynucleotidecan inhibit DNA replication or DNA transcription by, for example,interfering with the attachment of DNA or RNA polymerase to the promoterby binding to a transcriptional initiation site or a template. It caninterfere with processing of mRNA, poly(A) addition to mRNA ortranslation of mRNA by, for example, binding to regions of the RNAtranscript such as the ribosome binding site. It can promote inhibitorymechanisms of the cells, such as promoting RNA degradation via RNaseaction. The inhibitory polynucleotide can bind to the major groove ofthe duplex DNA to form a triple helical or “triplex” structure. Methodsof inhibition using inhibitory polynucleotides therefore encompass anumber of different approaches to altering expression of specific genesthat operate by different mechanisms. These different types ofinhibitory polynucleotide technology are described in C. Helene and J.Toulme, (1990) Biochim. Biophys. Acta., 1049:99-125.

Antisense polynucleotides can include deoxyribonucleotides orribonucleotides. They can be chemically modified so as to improvestability in the body. Properties of the polynucleotide can beengineered to impart stability (e.g., nuclease resistance), tighterbinding or the desired T_(m). See, e.g., International patentpublication No. 94/12633.

The general approach to constructing various polynucleotides useful ininhibitory polynucleotide therapy has been reviewed by A. R. Vander Krolet al. (1988), Biotechniques 6:958-976, and by C. A. Stein et al.,(1988) Cancer Res. (1988) 48:2659-2668. See also Oligodeoxynucleotides:Antisense Inhibitors of Gene Expression, Cohen, J. S., editor, MacMillanPress, London, pages 79-196 (1989), and Antisense RNA and DNA, (1988),D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. In certain embodiments inhibitory polynucleotides comprise aderivatized substituent which is substantially non-interfering withrespect to hybridization of the inhibitory polynucleotide to the targetpolynucleotide.

B. Antisense

This invention provides antisense polynucleotides capable ofspecifically hybridizing to a target sequence of Repro-EN-1.0 or IB1.Antisense polynucleotides are useful in vitro or in vivo to inhibit theactivity of Repro-EN-1.0 or IB1.

The antisense polynucleotides of this invention comprise an antisensesequence of at least 7 nucleotides that specifically hybridize to asequence from Repro-EN-1.0 or IB1 and, more particularly, mammalianRepro-EN-1.0 or IB1 and human Repro-EN-1.0 or IB1.

The antisense sequence can be between about 10 and about 50 nucleotidesor between about 15 and about 35 nucleotides. In other embodiments,antisense polynucleotides are polynucleotides of less than about 100nucleotides or less than about 200 nucleotides. Accordingly, a sequenceof the antisense polynucleotide can specifically hybridize to all orpart of the Repro-EN-1.0 or IB1, such as antisense polynucleotides tothe Repro-EN-1.0 or IB1 gene or its transcribed RNA. In one embodiment,the sequence of the polynucleotide contains within it the antisensesequence. In this case, the antisense sequence is contained within apolynucleotide of longer sequence. In another embodiment, the sequenceof the polynucleotide consists essentially of, or is, the antisensesequence. Thus, for example, the antisense polynucleotide can be apolynucleotide of less than about 50 nucleotides in a sequence thatspecifically hybridizes to the target sequence.

Generally, to assure specific hybridization, the antisense sequence issubstantially complementary to the target sequence in Repro-EN-1.0 orIB1. In certain embodiments, the antisense sequence is exactlycomplementary to the target sequence. The antisense polynucleotides mayinclude nucleotide substitutions, additions, deletions, ortranspositions, so long as specific binding to the relevant targetsequence corresponding to Repro-EN-1.0 mRNA or its gene, or IB1 mRNA orits gene, is retained as a functional property of the polynucleotide.

The antisense polynucleotide should be long enough to form a stableduplex but short enough, depending on the mode of delivery, toadminister in vivo, if desired. The minimum length of a polynucleotiderequired for specific hybridization to a target sequence depends onseveral factors, such as G/C content, positioning of mismatched bases(if any), degree of uniqueness of the sequence as compared to thepopulation of target polynucleotides, and chemical nature of thepolynucleotide (e.g., methylphosphonate backbone, polyamide nucleicacid, phosphorothioate, etc.), among others.

For general methods relating to antisense polynucleotides, see AntisenseRNA and DNA, (1988), D. A. Melton, Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). For a review of antisense therapy, see, e.g.,Uhlmann et al., Chem. Reviews, 90:543-584 (1990).

C. Ribozymes

Cleavage of Repro-EN-1.0 or IB1 can be induced by the use of ribozymesor catalytic RNA. In this approach, the ribozyme would contain eithernaturally occurring RNA (ribozymes) or synthetic nucleic acids withcatalytic activity. Bratty et al., (1992) Biochim. Biophys. Acta.,1216:345-59 (1993) and Denhardt, (1992) Ann. N.Y. Acad. Sci., 660:70-76describe methods for making ribozymes.

Unlike the antisense and other polynucleotides described above, whichbind to an RNA or a DNA, a ribozyme not only binds but also specificallycleaves and thereby potentially inactivates a target RNA. Such aribozyme can comprise 5′- and 3′-terminal sequences complementary to theRepro-EN-1.0 RNA or IB1 RNA.

Optimum target sites for ribozyme-mediated inhibition of activity can bedetermined as described by Sullivan et al., PCT patent publication No.94/02595 and Draper et al., PCT patent publication No. 93/23569. Asdescribed by Hu et al., PCT patent publication No. 94/03596, antisenseand ribozyme functions can be combined in a single polynucleotide. Uponreview of the RNA sequence of Repro-EN-1.0 and IB1, those in the artwill note that several useful ribozyme target sites are present andsusceptible to cleavage by, for example, a hammerhead motif ribozyme.

Such engineered ribozymes can be expressed in cells or can betransferred by a variety of means (e.g., liposomes, immunoliposomes,biolistics, direct uptake into cells, etc.). Other forms of ribozymes(group I intron ribozymes (Cech (1995) Biotechnology, 13; 323);hammerhead ribozymes (Edgington (1992) Biotechnology 10: 256) can beengineered on the basis of the disclosed Repro-EN-1.0 or IB1 sequenceinformation to catalyze cleavage of Repro-EN-1.0 RNA or IB1 RNA.Moreover, ribozymes can comprise one or more modified nucleotides ormodified linkages between nucleotides, as described above.

D. Other Inhibitory Polynucleotides

In addition to the antisense and ribozyme inhibitory polynucleotides,one can construct polynucleotides that will bind to duplex nucleic acideither in the folded RNA component or in the gene for the RNA component,forming a triple helix-containing or triplex nucleic acid to inhibitRepro-EN-1.0 or IB1 activity. Such polynucleotides of the invention areconstructed using the base-pairing rules of triple helix formation andthe nucleotide sequence of the RNA component (Cheng et al. (1988) J.Biol. Chem. 263: 15110; Ferrin and Camerini-Otero (1991) Science 354:1494; Ramdas et al. (1989) J. Biol. Chem. 264: 17395; Strobel et al.(1991) Science 254: 1639; Hsieh et al. (1990) op.cit.; Rigas et al.(1986) Proc. Natl. Acad. Sci. (U.S.A.) 83: 9591. Such polynucleotidescan block Repro-EN-1.0 or IB1 activity in a number of ways, including bypreventing transcription of the Repro-EN-1.0 or IB1 gene.

Typically, and depending on mode of action, the triplex-formingpolynucleotides of the invention comprise a sequence large enough toform a stable triple helix but small enough, depending on the mode ofdelivery, to administer in vivo.

E. Methods for Making Inhibitory Polynucleotides

Inhibitory polynucleotides can be made chemically or recombinantly.

1. Chemical Synthesis

Small inhibitory polynucleotides for direct delivery can be made bychemical synthesis. Chemically synthesized polynucleotides can be DNA orRNA, or can include nucleotide analogs or backbones that are not limitedto phosphodiester linkages.

2. Recombinant Production

For delivery into cells or for gene therapy methods, recombinantproduction of inhibitory polynucleotides through the use of expressionvectors is particularly useful. Accordingly, this invention alsoprovides expression vectors, e.g., recombinant polynucleotide moleculescomprising expression control sequences operatively linked to thenucleotide sequence encoding the inhibitory polynucleotide.

VII. Repro-EN-1.0 and IB1 Polypeptides

This invention also provides purified, recombinant Repro-EN-1.0 and IB1polypeptide, and Repro-EN-1.0 and IB1 analogs. Recombinant Repro-EN-1.0polypeptide includes the polypeptide whose amino acid sequence ispresented in SEQ ID NO:2, as well as allelic variants of it.Repro-EN-1.0 analogs include 1) immunogenic fragments of Repro-EN-1.0;2) homologs of Repro-EN-1.0 from other mammals (especially primates); 3)fragments of Repro-EN-1.0 comprising at least 5 consecutive amino acidsfrom the sequence of Repro-EN-1.0; 4) non-naturally occurringpolypeptides whose sequences are substantially identical toRepro-EN-1.0; and 5) fusion proteins comprising a Repro-EN-1.0 or aRepro-EN-1.0 analog fused to a second polypeptide moiety.

Repro-EN-1.0 polypeptide refers to native Repro-EN-1.0, the polypeptidewhose amino acid sequence is the amino acid sequence of SEQ ID NO:2, andto allelic variants of it. Polynucleotide molecules that encode allelicvariants of Repro-EN-1.0 are isolatable from endometrial cancer cellcDNA or genomic DNA and typically hybridize under stringent conditionsto the nucleotide sequence encoding Repro-EN-1.0 (SEQ ID NO: 1). Theycan be obtained by amplification using, e.g., PCR primers taken from thesequence of Repro-EN-1.0 described herein.

Repro-EN-1.0 polypeptides are useful as immunogens to elicit theproduction of anti-Repro-EN-1.0 antibodies, as affinity capturemolecules to isolate such antibodies from a mixture, and as controls indiagnostic methods aimed at detecting Repro-EN-1.0 in a sample.

Immunogenic Repro-EN-1.0 analogs are polypeptides having a sequence ofat least 5 amino acids selected from native Repro-EN-1.0 and which, whenpresented to an animal as an immunogen, elicit a humoral orcell-mediated immune response. This includes polypeptides comprising anamino acid sequence which is an epitope from Repro-EN-1.0, such asimmunogenic fragments of Repro-EN-1.0. Repro-EN-1.0 protein analogsoptionally are in isolated form. Persons skilled in the art are familiarwith methods of identifying probable epitopes of a protein. For example,polypeptide fragments most likely to elicit an immune response againstthe native protein are those that exist on the surface of the nativeprotein. Portions of a protein on the surface tend to includehydrophilic amino acids. Such regions can be identified by inspection orwith available software. In one embodiment immunogenic polypeptide is apolypeptide comprising a sequence of at least 5 consecutive hydrophilicamino acids, or at least five hydrophilic amino acids in a series ofeight consecutive amino acids. Repro-EN-1.0 contains long hydrophilicstretches. Examples of such polypeptides include those comprising thesequences:

KTPSAEERR, (SEQ ID NO: 7) RARPESER, (SEQ ID NO: 8) RMSDMLSR (SEQ ID NO:9) or NEKLSPKPG. (SEQ ID NO: 10)

The cDNA encoding Repro-EN-1.0 of SEQ ID NO: 1 was discovered byscreening an expression library of cDNA from an endometrial carcinomacell line with serum pooled from subjects diagnosed with endometriosis.Therefore, polypeptides comprising an epitope of Repro-EN-1.0 can beidentified by screening with such serum. Preferably, the test serum is aserum pooled from several subjects positively diagnosed withendometriosis. At least one epitope of Repro-EN-1.0 exists in a fragmentof 567 amino acids from the carboxy-terminus of the molecule (aminoacids 293-860 of SEQ ID NO:2). At least one epitope of IB1 exists in afragment of 644 amino acids from the carboxy terminus of the molecule(amino acids 293-937 of SEQ ID NO:4). Immunogenic fragments are useful,for example, to detect the presence of antibodies against Repro-EN-1.0or IB1 in patient serum samples. This test is useful in diagnosisbecause the presence of such antibodies is a diagnostic marker forendometriosis.

Fragments of Repro-EN-1.0 or IB1 include those having at least 5 aminoacids, at least 10 amino acids, at least 50 amino acids, at least 100amino acids or at least 200 amino acids in a sequence from Repro-EN-1.0or IB1. Fragments are useful as immunogens to produce an iminuneresponse against Repro-EN-1.0 or IB1 in the production of antibodies.Alternatively, fragments having appropriate amino acid motifs are usefulas agretopes to bind with MHC molecules. Such complexes are useful ininducing anergy against Repro-EN-1.0 or IB1.

Non-naturally occurring analogs of Repro-EN-1.0 or IB1 have at least 90%sequence identity with Repro-EN-1.0 or IB1. They can be made by, forexample, introducing conservative amino acid substitutions into thesequence of Repro-EN-1.0 or IB1. Such molecules are useful as decoys oras active analogs.

Homologs of Repro-EN-1.0 or IB1 from other animals generally havesequences that are substantially identical to that of SEQ ID NO:1 or SEQID NO:3. Genomic DNA or cDNA encoding them can be identified throughscreening libraries under stringent hybridization conditions using aRepro-EN-1.0 or IB1 probe of this invention.

Fusion proteins include a fragment of Repro-EN-1.0 or IB1 fused to asecond polypeptide moiety at the carboxy or amino terminus. The secondpolypeptide can function, for example, as a detectable label. Suchmarkers include fluorescent protein, enzyme marker of protein from atwo-hybrid system.

Repro-EN-1-0 or IB1 and analogs are most easily produced recombinantly,as described herein. Recombinant Repro-EN-1.0 or IB1 can be purified byaffinity purification. In one method, recombinant Repro-EN-1.0 or IB1analogs comprise a polyhistidine tag. The protein is purified on anickel-chelate affinity matrix. In another method, Repro-EN-1.0 or IB1is purified using an affinity matrix carrying anti-Repro-EN-1.0 or IB1antibodies.

VIII. Antibodies and Hybridomas

In one aspect this invention provides a composition comprising anantibody that specifically binds Repro-EN-1.0 or IB1 polypeptides.Antibodies preferably have affinity of at least 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸M⁻¹, or 10⁹ M⁻¹. This invention contemplates both polyclonal andmonoclonal antibody compositions.

In one embodiment this invention provides immunotoxins againstRepro-EN-1.0- or IB1-expressing cells. Immunotoxins are antibodies andthe like as described herein coupled to a compound, e.g., a toxin, thatis toxic to a target cell. Toxins can include, for example, radioactiveisotopes, ricin, cisplatin, antisense molecules, Diphtheria toxin,Pseudomonas exotoxin A or Bacillus anthraces protective antigen.Immunotoxins bind to cancer cells that express Repro-EN-1.0 or IB1 andkill them. They are useful in the therapeutic methods of this invention.The antibodies of the invention have many uses. For example, suchantibodies are useful for detecting Repro-EN-1.0 or IB1 polypeptides inimmunoassays. The antibodies also can be used to screen expressionlibraries for particular expression products such as mammalianRepro-EN-1.0 or IB1. These are useful for detecting or diagnosingvarious pathological conditions related to the presence of therespective antigens. Usually the antibodies in such a procedure arelabeled with a moiety allowing easy detection of presence of antigen byantibody binding. Antibodies raised against Repro-EN-1.0 or IB1 can alsobe used to raise anti-idiotypic antibodies.

A. Production of Antibodies

A number of immunogens are used to produce antibodies that specificallybind Repro-EN-1.0 or IB1 polypeptides. Full-length Repro-EN-1.0 or IB1is a suitable immunogen. Typically, the immunogen of interest is apeptide of at least about 3 amino acids, more typically the peptide is 5amino acids in length, preferably, the fragment is 10 amino acids inlength and more preferably the fragment is 15 amino acids in length orgreater. The peptides can be coupled to a carrier protein (e.g., as afusion protein), or are recombinantly expressed in an immunizationvector. Antigenic determinants on peptides to which antibodies bind aretypically 3 to 10 amino acids in length. Naturally occurringpolypeptides are also used either in pure or impure form.

Recombinant polypeptides are expressed in eukaryotic or prokaryoticcells and purified using standard techniques. The polypeptide, or asynthetic version thereof, is then injected into an animal capable ofproducing antibodies. Either monoclonal or polyclonal antibodies can begenerated for subsequent use in immunoassays to measure the presence andquantity of the polypeptide.

Methods for producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen, preferably a purified polypeptide, apolypeptide coupled to an appropriate carrier (e.g., GST, keyhole limpethemanocyanin, etc.), or a polypeptide incorporated into an immunizationvector such as a recombinant vaccinia virus (see, U.S. Pat. No.4,722,848) is mixed with an adjuvant and animals are immunized with themixture. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the polypeptide of interest. When appropriately high titers ofantibody to the immuriogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the polypeptide is performed wheredesired. See, e.g., Coligan (1991) Current Protocols in ImmunologyWiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: A LaboratoryManual Cold Spring Harbor Press, NY.

Antibodies, including binding fragments and single chain recombinantversions thereof, against predetermined fragments of Repro-EN-1.0 or IB1proteins are raised by immunizing animals, e.g., with conjugates of thefragments with carrier proteins as described above.

Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies are screened for binding to normal ormodified polypeptides, or screened for agonistic or antagonisticactivity, e.g., activity mediated through Repro-EN-1.0 or IB1. In someinstances, it is desirable to prepare monoclonal antibodies from variousmammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arefound in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4thed.) Lange Medical Publications, Los Altos, Calif., and references citedtherein; Harlow and Lane, Supra; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andKohler and Milstein (1975) Nature 256: 495-497.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al. (1989) Science246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546.

Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S.Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA86: 10029-10033.

Frequently, the polypeptides and antibodies will be labeled by joining,either covalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Thus, an antibody used for detecting an analyte can bedirectly labeled with a detectable moiety, or may be indirectly labeledby, for example, binding to the antibody a secondary antibody that is,itself directly or indirectly labeled.

The antibodies of this invention are also used for affinitychromatography in isolating Repro-EN-1.0 or IB1 proteins. Columns areprepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysateis passed through the column, washed, and treated with increasingconcentrations of a mild denaturant, whereby purified Repro-EN-1.0 orIB1 polypeptides are released.

An alternative approach is the generation of humanized immunoglobulinsby linking the CDR regions of non-human antibodies to human constantregions by recombinant DNA techniques. See Queen et al., U.S. Pat. No.5,585,089.

A further approach for isolating DNA sequences which encode a humanmonoclonal antibody or a binding fragment thereof is by screening a DNAlibrary from human B cells according to the general protocol outlined byHuse et al., Science 246:1275-1281 (1989) and then cloning andamplifying the sequences which encode the antibody (or binding fragment)of the desired specificity. The protocol described by Huse is renderedmore efficient in combination with phage display technology. See, e.g.,Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047. Phagedisplay technology can also be used to mutagenize CDR regions ofantibodies previously shown to have affinity for Repro-EN-1.0 or IB1protein receptors or their ligands. Antibodies having improved bindingaffinity are selected.

In another embodiment of the invention, fragments of antibodies againstRepro-EN-1.0 or IB1 protein or protein analogs are provided. Typically,these fragments exhibit specific binding to the Repro-EN-1.0 proteinreceptor similar to that of a complete immunoglobulin. Antibodyfragments include separate heavy chains, light chains Fab, Fab′ F(ab′)₂and Fv. Fragments are produced by recombinant DNA techniques, or byenzymic or chemical separation of intact immunoglobulins.

IX. Methods for Detecting Repro-EN-1.0 and IB1 Polypeptides

Repro-EN-1.0 or IB1 polypeptides can be identified by any methods knownin the art. In one embodiment, the methods involve detecting thepolypeptide with a ligand that specifically recognizes the polypeptide(e.g., an immunoassay). The antibodies of the invention are particularlyuseful for specific detection of Repro-EN-1.0 or IB1 polypeptides. Avariety of antibody-based detection methods are known in the art. Theseinclude, for example, radioimmunoassay, sandwich immunoassays (includingELISA), immunofluorescent assays, western blot, affinity chromatography(affinity ligand bound to a solid phase), and in situ detection withlabeled antibodies. Another method for detecting Repro-EN-1.0 or IB1polypeptides involves identifying the polypeptide according to its massthrough, for example, gel electrophoresis, mass spectrometry or HPLC.Subject samples can be taken from any number of appropriate sources,such as saliva, peritoneal fluid, blood or a blood product (e.g.,serum), urine, tissue biopsy (e.g., lymph node tissue), etc.

a. Immunoassays

The present invention also provides methods for detection ofRepro-EN-1.0 or IB1 polypeptides employing one or more anti-Repro-EN-1.0or IB1 antibody reagents (i.e., immunoassays). A number of wellestablished immunological binding assay formats suitable for thepractice of the invention are known (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). See, e.g., METHODS INCELL BIOLOGY VOLUME 37: ANTIBODIES IN CELL BIOLOGY, Asai, ed. AcademicPress, Inc. New York (1993); BASIC AND CLINICAL IMMUNOLOGY 7th Edition,Stites & Ten, eds. (1991); Harlow and Lane, supra [e.g., Chapter 14],and Ausubel et al., supra, [e.g., Chapter 11]. Typically, immunologicalbinding assays (or immunoassays) utilize a “capture agent” tospecifically bind to and, often, immobilize the analyte to a solidphase. In one embodiment, the capture agent is a moiety thatspecifically binds to a Repro-EN-1.0 or IB1 polypeptide or subsequence,such as an anti-Repro-EN-1.0 or anti-IB1 antibody.

Usually the Repro-EN-1.0 or IB1 polypeptide being assayed is detecteddirectly or indirectly using a detectable label. The particular label ordetectable group used in the assay is usually not a critical aspect ofthe invention, so long as it does not significantly interfere with thespecific binding of the antibody or antibodies used in the assay. Thelabel may be covalently attached to the capture agent (e.g., ananti-Repro-EN-1.0 or anti-IB1 antibody), or may be attached to a thirdmoiety, such as another antibody, that specifically binds to theRepro-EN-1.0 polypeptide.

The present invention provides methods and reagents for competitive andnoncompetitive immunoassays for detecting Repro-EN-1.0 or IB1polypeptides. Non-competitive immunoassays are assays in which theamount of captured analyte (in this case Repro-EN-1.0 or IB1) isdirectly measured. One such assay is a two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on the Repro-EN-1.0 or IB1 protein. See, e.g.,Maddox et al., 1983, J. Exp. Med., 158:1211 for background information.In one preferred “sandwich” assay, the capture agent (e.g., ananti-Repro-EN-1.0 or anti-IB1 antibody) is bound directly to a solidsubstrate where it is immobilized. These immobilized antibodies thencapture any Repro-EN-1.0 or IB1 protein present in the test sample. TheRepro-EN-1.0 or IB1 polypeptide thus immobilized can then be labeled,i.e., by binding to a second anti-Repro-EN-1.0 or IB1 antibody bearing alabel. Alternatively, the second anti-Repro-EN-1.0 or IB1 antibody maylack a label, but be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond antibody alternatively can be modified with a detectable moiety,such as biotin, to which a third labeled molecule can specifically bind,such as enzyme-labeled streptavidin.

In competitive assays, the amount of Repro-EN-1.0 or IB1 protein presentin the sample is measured indirectly by measuring the amount of an added(exogenous) Repro-EN-1.0 or IB1 displaced (or competed away) from acapture agent (e.g., anti-Repro-EN-1.0 or anti-IB1 antibody) by theRepro-EN-1.0 or IB1 protein present in the sample.

A hapten inhibition assay is another example of a competitive assay. Inthis assay Repro-EN-1.0 or IB1 protein is immobilized on a solidsubstrate. A known amount of anti-Repro-EN-1.0 or anti-IB1 antibody isadded to the sample, and the sample is then contacted with theimmobilized Repro-EN-1.0 or IB1 protein. In this case, the amount ofanti-Repro-EN-1.0 or anti-IB1 antibody bound to the immobilizedRepro-EN-1.0 or IB1 protein is inversely proportional to the amount ofRepro-EN-1.0 protein present in the sample. The amount of immobilizedantibody may be detected by detecting either the immobilized fraction ofantibody or the fraction of the antibody that remains in solution. Inthis aspect, detection may be direct, where the antibody is labeled, orindirect where the label is bound to a molecule that specifically bindsto the antibody as described above.

b. Other Antibody-Based Assay Formats

The invention also provides reagents and methods for detecting andquantifying the presence of Repro-EN-1.0 or IB1 polypeptide in thesample by using an immunoblot (Western blot) format. Another immunoassayis the so-called “lateral flow chromatography.” In a non-competitiveversion of lateral flow chromatography, a sample moves across asubstrate by, e.g., capillary action, and encounters a mobile labeledantibody that binds the analyte forming a conjugate. The conjugate thenmoves across the substrate and encounters an immobilized second antibodythat binds the analyte. Thus, immobilized analyte is detected bydetecting the labeled antibody. In a competitive version of lateral flowchromatography a labeled version of the analyte moves across the carrierand competes with unlabeled analyte for binding with the immobilizedantibody. The greater the amount of the analyte in the sample, the lessthe binding by labeled analyte and, therefore, the weaker the signal.See, e.g., May et al., U.S. Pat. No. 5,622,871 and Rosenstein, U.S. Pat.No. 5,591,645.

c. Solid Phases: Substrates, Solid Supports, Membranes, Filters

As noted supra, depending upon the assay, various components, includingthe antigen, target antibody, or anti-Repro-EN-1.0 or anti-IB1 antibody,may be bound to a solid surface or support (i.e., a substrate, membrane,or filter paper). Many methods for immobilizing biomolecules to avariety of solid surfaces are known in the art. For instance, the solidsurface may be a membrane (e.g., nitrocellulose), a microtiter dish(e.g., PVC, polypropylene, or polystyrene), a test tube (glass orplastic), a dipstick (e.g. glass, PVC, polypropylene, polystyrene,latex, and the like), a microcentrifuge tube, or a glass or plasticbead. The desired component may be covalently bound or noncovalentlyattached through nonspecific bonding.

A wide variety of organic and inorganic polymers, both natural andsynthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers which formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

d. Mass Spectrometry

The mass of a molecule frequently can be used as an identifier of themolecule. Therefore, methods of mass spectrometry can be used toidentify a protein analyte. Mass spectrometers can measure mass bydetermining the time required for an ionized analyte to travel down aflight tube and to be detected by an ion detector.

One method of mass spectrometry for proteins is matrix-assisted laserdesorption/ionization mass spectrometry (“MALDI”). In MALDI the analyteis mixed with an energy absorbing matrix material that absorbs energy ofthe wavelength of a laser and placed on the surface of a probe. Uponstriking the matrix with the laser, the analyte is desorbed from theprobe surface, ionized, and detected by the ion detector. See, forexample, Hillenkamp et al., U.S. Pat. No. 5,118,937.

Other methods of mass spectrometry for proteins are described inHutchens and Yip, U.S. Pat. No. 5,719,060. In one such method referredto as Surfaces Enhanced for Affinity Capture (“SEAC”) a solid phaseaffinity reagent that binds the analyte specifically ornon-specifically, such as an antibody or a metal ion, is used toseparate the analyte from other materials in a sample. Then the capturedanalyte is desorbed from the solid phase by, e.g., laser energy,ionized, and detected by the detector.

e. Assay Combinations

The diagnostic and prognostic assays described herein can be carried outin various combinations and can also be carried out in conjunction withother diagnostic or prognostic tests. For example, when the presentmethods are used to diagnose endometriosis, the presence of aRepro-EN-1.0 or IB1 polypeptide can be used to determine the stage ofthe disease. Tests that may provide additional information includemicroscopic analysis of biopsy samples, detection of antigens (e.g.,cell-surface markers) associated with endometriosis (e.g., usinghistocytochemistry, FACS, or the like).

X. Diagnostic, Monitoring and Prognostic Methods

A. Methods of Diagnosing Endometriosis

We have detected circulating antibodies against Repro-EN-1.0 or IB1 inthe blood of women diagnosed with endometriosis. This supports the ideathat endometriosis has an autoimmune component. Further, Repro-EN-1.0 orIB1 and auto-antibodies against Repro-EN-1.0 or IB1 represent twotargets in the diagnosis of endometriosis.

Repro-EN-1.0 or IB1 that is shed into the peritoneal fluid of women withendometriosis is useful in methods of diagnosing endometriosis. Thesemethods include detecting Repro-EN-1.0 or IB31 in a biological sample ofa subject. Suitable samples include, without limitation, saliva, bloodor a blood product (e.g., serum), urine, menstrual fluid, vaginalsecretion and, in particular, peritoneal fluid. Repro-EN-1.0 or IB1 canbe detected by any of the methods described herein. Any detection ofRepro-EN-1.0 or IB1 above a normal range is a positive sign in thediagnosis of endometriosis.

In another aspect, this invention provides methods for diagnosingendometriosis in a subject by detecting in a sample from the subject adiagnostic amount of an antibody that specifically binds to Repro-EN-1.0or IB1 polypeptide. Suitable patient samples include, withoutlimitation, saliva, blood or a blood product (e.g., serum), peritonealfluid, urine, menstrual fluid, vaginal secretion. The antibodies can bedetected by any of the methods for detecting proteins described herein.However, sandwich type assays are particularly useful. In one version,all antibodies are captured onto a solid phase, for example usingprotein A, and antibodies specific for Repro-EN-1.0 or IB1 are detectedusing a directly or indirectly labeled Repro-EN-1.0 or IB1 orpolypeptide fragment of it having an epitope of Repro-EN-1.0 or IB1. Inanother version of the assay, Repro-EN-1.0 or IB1 or an antigenicfragment of it can be used as the capture molecule and capturedantibodies can be detected.

While the detection of antibodies, in general, against Repro-EN-1.0 orIB1 is a positive sign of endometriosis, IgE an IgG₄ class antibodiesare particularly specific and sensitive for the diagnosis ofendometriosis. Therefore, in one embodiment, the diagnostic methodinvolves specifically detecting IgE or IgG₄ antibodies that specificallyrecognize Repro-EN-1.0 or IB1. Anti-human IgE antibodies and anti-humanIgG₄ antibodies can be easily bought or made.

1. In Vivo Diagnosis

In another method of the invention, endometriosis can be diagnosed invivo. The methods involve detecting Repro-EN-1.0 or IB1 in the body,e.g., in the peritoneum. In general, any conventional method forvisualizing diagnostic imaging can be used.

In one method for diagnosing endometriosis, detection is performed bylaparoscopy. A ligand specific for Repro-EN-1.0 or IB1 is introducedinto the subject at the site of a suspected lesion and binding isdetected using the laparoscope. Alternatively, the binding can bedetected by, for example, magnetic resonance imaging (MRI) or electronspin resonance (ESR). Usually gamma-emitting and positron-emittingradioisotopes are used for camera imaging and paramagnetic isotopes areused for magnetic resonance imaging. Any amount of binding abovebackground is a positive sign of endometriosis. Persons of skill in theart recognize that not every positive sign results in a definitivediagnosis of a disease.

Endometriotic lesions can be removed surgically. However, lesions may betiny, and difficult to identify by eye. This invention takes advantageof Repro-EN-1.0 or IB1 as marker for endometriosis by providing a methodto identify and remove endometriotic lesions. The method involvesidentifying endometriotic lesions in situ using a labeled probe directedto Repro-EN-1.0 or IB1. Then, the lesions are removed surgically.

In the practice of this method, a probe is provided. The probe binds toRepro-EN-1.0 or IB1 and is labeled with a detectable marker that can bedetected in a surgical procedure. In particular, the probe can be anantibody that specifically binds Repro-EN-1.0 or IB1. Preferred labelsthat can be detected during surgery are radioactive labels andfluorescent labels. Radioactive labels can be detected with the use of,e.g., a Geiger counter. Fluorescent labels, such as FITC, can bedetected using, e.g., a D-Light-System (Storz, Tuttlingen Germany).

Surgery can proceed as follows. The labeled probe is introduced into theperitoneum of the patient for a time sufficient for the label to bind toendometriotic lesions. Unbound labeled probe is washed out. Then,endometriotic lesions are identified using a suitable detector. Forexample, in laparoscopic surgery, a Geiger counter may be introducedthrough the incision. Radioactive (“hot”) spots indicate bound labeledand, therefore, an endometriotic lesion. These lesions are then removedfrom the patient.

B. Methods of Diagnosing Cancer

Repro-EN-1.0 or IB1 is up-regulated in breast cancer cells, uterinecancer cells, and prostate cancer cells. Therefore, Repro-EN-1.0 or IB1is a marker for these pathologic conditions. Accordingly, the methodsdescribed herein for detecting Repro-EN-1.0 or IB1 polynucleotides orRepro-EN-1.0 or IB1 polypeptides in a sample are useful in methods fordiagnosing these cancers, monitoring their progress or treatment, anddetermining patient prognosis. The methods of the present inventionallow cancerous conditions to be detected with increased confidence andat an earlier stage, before cells are detected as cancerous based onpathological characteristics. It is, of course, understood bydiagnosticians that diagnostic tests are measured by their degree ofspecificity and sensitivity. Tests which are not perfectly specific orsensitive are, nevertheless, useful in diagnosis because they provideuseful information which, in combination with other evidence, canprovide a definitive diagnosis or indicate a course of treatment.

Methods for diagnosis involve determining a diagnostic amount ofRepro-EN-1.0 or IB1 (e.g., mRNA, cDNA or polypeptide) in a patientsample and comparing that amount with a normal range (e.g., a controlamount) expected to be found in the sample. The samples used todetermine the normal range of Repro-EN-1.0 or IB1 can be normal samplesfrom the individual to be tested, or normal samples from otherindividuals not suffering from the disease condition.

A variety of patient samples can be used in the methods of theinvention. For example, cell extracts, cultured cells, or tissue samplesprovide convenient samples for use with the methods of the invention.The methods of the invention can use samples either in solution orextracts, for example, with RT-PCR, or samples such as tissue sectionsfor in situ methods of detection. Samples can also be obtained fromsources such as fine-needle biopsies, e.g., from breast, uterus orprostate; cellular materials; whole cells; tissue and cell extracts; RNAextracted from tissue and cells and histological sections of tissue.

Methods for monitoring the course of a cancer with which Repro-EN-1.0 orIB1 is associated involve determining the amount of Repro-EN-1.0 or IB1in a sample at a first and second time. The times can be during routinephysical examinations or during a course of treatment for the cancer. Ascancer appears and/or progresses, the amount of Repro-EN-1.0 or IB1 in asample is expected to increase. Regression or cure of the cancer areaccompanied by a decrease or elimination of Repro-EN-1.0 or IB1 in asample.

The diagnostic and prognostic methods can also be carried out inconjunction with other diagnostic or prognostic tests. In someinstances, such combination tests can provide useful informationregarding the progression of a disease, although the present methods fortesting for Repro-EN-1.0 or IB1 provide much useful information in thisregard.

Another diagnostic method of the invention involves the administrationto a subject of a labeled composition that specifically binds to cellsbearing Repro-EN-1.0, or IB1 such as labelled antibodies. Then, thelocalization of the label is determined by any of the known radiologicmethods. Any conventional method for visualizing diagnostic imaging canbe used. Usually gamma- and positron-emitting radioisotopes are used forcamera imaging and paramagnetic isotopes are used for MRI.

C. Methods of Diagnosing Chromosomal Changes

The Repro-EN-1.0 or IB1 gene is located on chromosome 1. A translocationat this site can result in alteration of Repro-EN-1.0 or IB1 activity,such as activated transcription or changed function. Chromosomaltranslocations in the vicinity of the Repro-EN-1.0 or IB1 gene can bedetected by hybridizing a labeled probe of this invention to achromosome spread. A translocation, duplication or deletion can beidentified by aberrant hybridization patterns compared to normal. Suchtests are useful in detecting genetic abnormalities such as familialdisposition to breast, uterine or prostate cancer, or early onset of thedisease. A method for fluorescent in situ hybridization of chromosomesis provided in the Examples.

The present invention also provides for kits for performing thediagnostic and prognostic method of the invention. Such kits include apolynucleotide probe or primer, or an antibody specific for Repro-EN-1.0or IB1 and instructions to use the reagents to detect Repro-EN-1.0 orIB1 in a patient sample.

XI. Methods for Inhibiting Repro-EN-1.0 or IB1 Expression or Activityand of Treating Cancer

Inhibiting Repro-EN-1.0 or IB1 expression or activity in a breast,uterine or prostate cancer cell can alter the rate of growth oraggressiveness of the cancer. Inhibiting Repro-EN-1.0 or IB1 expressionor activity is useful in vivo in the prophylactic and therapeutictreatment of prostate cancer or other conditions involving Repro-EN-1.0or IB1 expression. Accordingly, this invention provides methods forinhibiting Repro-EN-1.0 or IB1 expression or activity. The methodsinvolve contacting a prostate cancer cell, in vitro or in vivo, with aninhibitory polynucleotide, an immunotoxin or another compound thatinhibits Repro-EN-1.0 or IB1 expression or activity.

A. Delivery of Inhibitory Polynucleotides

This invention contemplates a variety of means for delivering aninhibitory polynucleotide to a subject including, for example, directuptake of the molecule by a cell from solution, facilitated uptakethrough lipofection (e.g., liposomes or immunoliposomes),particle-mediated transfection, and intracellular expression from anexpression cassette having an expression control sequence operablylinked to a nucleotide sequence that encodes the inhibitorypolynucleotide. Methods useful for delivery of polynucleotides fortherapeutic for therapeutic purposes are described in Inouye et al.,U.S. Pat. No. 5,272,065.

B. Pharmaceutical Compositions and Treatment

Agents, such as inhibitory polynucleotides, immunotoxins or othercompounds that inhibit Repro-EN-1.0 or IB1 expression or activitypreferably are delivered in pharmaceutical compositions comprising theagent and a pharmaceutically acceptable carrier. The agent can beadministered by any route that gives it access to cells expressingRepro-EN-1.0 or IB1, for example, prostate tumor cells. This includes,for example, aqueous solutions for enteral, parenteral or transmucosaladministration, e.g., for intravenous administration, as tonics andadministration to mucous or other membranes as, for example, nose or eyedrops; solid and other non-aqueous compositions for enteral ortransdermal delivery, e.g., as pills, tablets, powders or capsules;transdermal or transmucosal delivery systems for topical administration,and aerosols or mists for delivery by inhalation. One advantage ofdelivery by a mode that is easy to administer, e.g., enteral or byintravenous or intramuscular injection is that such modes mimic possiblemodes of delivery should the agent be formulated as a pharmaceutical.

In one embodiment, the pharmaceutical composition is in the form of aunit dose which contains a pharmacologically effective amount of theRepro-EN-1.0 or IB1 inhibitory compound. The unit dose, taken as part ofa therapeutic regimen, results in inhibition of growth of prostatecancer cells. Thus, the pharmaceutical compositions of the invention,whatever the form, are administered in a pharmacologically effectiveamount to the subject.

The amount of the pharmaceutical composition delivered, the mode ofadministration and the time course of treatment are at the discretion ofthe treating physician. Prophylactic treatments are indicated forpersons at higher than average risk of getting prostate cancer, breastcancer or uterine cancer. For example, persons with elevated PSA, PAP(prostate acid phosphatase) or PSP (prostate specific protein) levelsare at increased risk of prostate cancer. Persons who have the BRCA1 orBRCA2 genes are at increased risk of breast cancer.

XII. Methods of Inhibiting an Immune Response Against Repro-EN-1.0 orIB1

Women with endometriosis exhibit auto-antibodies against Repro-EN-1.0.or IB1. This fact supports the idea that endometriosis involves anauto-immune response. Thus, this invention provides methods useful forinhibiting an immune response against Repro-EN-1.0 or IB1. In oneembodiment, the methods include suppressing the immune system in personswith endometriosis. This includes, for example, the administration ofimmunosuppressive drugs such as anti-histamines, anti-inflammatoriessuch as steroids, or cyclosporin or anti-idiotypic antibodies thatrecognize auto-antibodies against Repro-EN-1.0 or IB31.

In one embodiment of the invention, the immune response can bediminished by the eliciting in the subject anti-idiotypic antibodiesagainst auto-antibodies that specifically recognize Repro-EN-1.0 or IB1.Anti-idiotypic antibodies are produced by immunizing the subject withantibodies against Repro-EN-1.0 or IB1. The amount of delivery to elicitan immune response can be about 1 μg to about 500 μg given with one ormore booster administrations over about six weeks. Anti-idiotypicantibodies bind to the antigen binding site of the Repro-EN-1.0 or IB1antibodies, thereby blocking their function.

In another embodiment of the invention the method involves inducinganergy in T cells involved in a humoral or cell-mediated immune responseagainst Repro-EN-1.0 or IB1. Anergy can be induced by administering to asubject an MHC-peptide complex that comprises an MHC molecule coupled toa peptide epitope from Repro-EN-1.0 or IB1.

MHC Class I and MHC Class II molecules bind peptides having particularamino acid motifs in binding pockets located at the amino-terminus ofthe molecules. The MHC Class II molecule is a dimer formed from an alphaand a beta chain. The binding pocket is formed from portions of bothchains. However, a single beta chain suffices to bind a peptide havingthe appropriate amino acid motif.

MHC-peptide complexes are formed by contacting a peptide having theappropriate motif with an isolated MHC Class II molecule. Alternatively,the complexes can be formed by creating fusion proteins containing boththe MHC molecule and the polypeptide epitope.

Methods of inducing anergy involve administering an isolated MHC-peptidecomplex to an individual suffering from the auto-immune disease.Isolated complexes are complexes that do not exist anchored onto a cellsurface. MHC-peptide complexes and methods of using them to induceanergy are described in, for example, U.S. Pat. Nos. 5,468,481 (S. D.Sharma et al.) and 5,734,023 (B. Nag et al.).

MHC Class II molecules bind peptides having particular amino acid motifswell known in the art. HLA-A1 binding motif includes a first conservedresidue of T, S or M, a second conserved residue of D or E, and a thirdconserved residue of Y. Other second conserved residues are A, S or T.The first and second conserved residues are adjacent and are preferablyseparated from the third conserved residue by 6 to 7 residues. A secondmotif consists of a first conserved residue of E or D and a secondconserved residue of Y where the first and second conserved residues areseparated by 5 to 6 residues. The HLA-A3.2 binding motif includes afirst conserved residue of L, M, I, V, S, A, T and F at position 2 and asecond conserved residue of K, R or Y at the C-terminal end. Other firstconserved residues are C, G or D and alternatively E. Other secondconserved residues are H or F. The first and second conserved residuesare preferably separated by 6 to 7 residues. The HLA-A11 binding motifincludes a first conserved residue of T or V at position 2 and aC-terminal conserved residue of K. The first and second conservedresidues are preferably separated by 6 or 7 residues. The HLA-A24.1binding motif includes a first conserved residue of Y, F or W atposition 2 and a C terminal conserved residue of F, I, W, M or L. Thefirst and second conserved residues are preferably separated by 6 to 7residues.

This invention also provides a peptide comprising a linear epitopederived from the Repro-EN-1.0 or IB1 which specifically binds to an MHCmolecule. In certain embodiments, the peptide has between 8 and 12 aminoacids and the linear epitope has a Class I MHC molecule binding motif.

The following chart provides portions of the amino acid sequence ofRepro-EN-1.0 (SEQ ID NO:2). Amino acid numbers are indicated. Bracketedbars over the amino acid sequence indicate vertebrate MHC Class I or MHCClass II binding motifs. Amino acid numbers are indicated. Peptides ofabout 8-15 amino acids in length that include these motifs, includingpeptides whose entire amino acid sequence is selected from the sequenceof Repro-EN-1.0, bind to MHC molecules and can be used to induce acell-mediated or humoral immune response against Repro-EN-1.0. Most ofthe sequence of Repro-EN-1.0 is exposed on the protein surface and iscapable of eliciting an immune response.

This invention also provides a pharmaceutical composition capable ofinducing anergy against Repro-EN-1.0 comprising a pharmaceuticallyacceptable carrier and an effective amount of an MHC-peptide complex ofthis invention. The complex is capable of inducing anergy in Class IMHC-restricted cytotoxic T-lymphocytes or Class II MHC-restricted immuneresponse against cells expressing Repro-EN-1.0.

Repro-EN-1.0 includes many amino acid binding motifs for MHC Class I andMHC Class II. These motifs are provided in Table 1. The amino acidsequence numbers are provided and the motifs are indicated with bars.

TABLE 1

XIII. Transgenic Non-Human Animals

This invention also provides non-human mammals transgenic forRepro-EN-1.0 and IB1. As used herein, “animal transgenic forRepro-EN-1.0 or IB1” refers to an animal, in particular a mammal, whosegerm cells (i.e., oocytes or sperm), at least, comprise a recombinantnucleic acid molecule comprising expression control sequencesoperatively linked to a nucleic acid sequence encoding Repro-EN-1.0 orIB1. Such animals are useful, for example, as models in the study ofendometriosis, spontaneous abortion and disease pathways.

In one embodiment, the expression control sequences are not naturallyfound operatively linked to Repro-EN-1.0. In one embodiment, therecombinant nucleic acid comprises a non-native Repro-EN-1.0 codingsequence, i.e., a Repro-EN-1.0 sequence that the species does notproduce in nature. In one embodiment, the Repro-EN-1.0 is a humanRepro-EN-1.0. In another embodiment, the expression control sequencesare non-native expression control sequences introduced into the germcells so as to recombine with the naturally occurring gene and controlits expression. Particularly useful transgenic mammals of this inventioninclude rabbits and rodents such as mice.

The transgenic animals of this invention are produced, for example, byintroducing the recombinant nucleic acid molecule into a fertilized eggor embryonic stem (ES) cell, typically by microinjection,electroporation, lipofection, particle-mediated gene transfer. Thetransgenic animals express the heterologous nucleotide sequence intissues depending upon whether the promoter is inducible by a signal tothe cell, or is constitutive. Transgenic animals can be bred withnon-transgenic animals to produce transgenic animals with mixedcharacteristics.

XIV. Methods for Screening for Compounds that Regulate Expression ofRepro-EN-1.0 or IB1

Compounds that regulate the expression of Repro-EN-1.0 and IB1 arecandidates as therapeutic agents in the treatment of breast, uterine orprostate cancer. This invention provides methods for determining whethera compound regulates (e.g., activates or inhibits) expression ofRepro-EN-1.0 or IB1.

Methods for determining whether a compound regulates Repro-EN-1.0 or IB1expression involve administering to a cell or a test animal having anexpressible Repro-EN-1.0 or IB1 gene with the compound, and determiningwhether expression Repro-EN-1.0 or IB1 is altered. In one embodiment,the methods involve administering the compound to a culture comprisingthe cell or to a test animal that has cells expressing Repro-EN-1.0 orIB1, measuring the amount of the Repro-EN-1.0 or IB1 polynucleotide orpolypeptide in a sample from the culture or the animal, and determiningwhether the measured amount is different than the amount in a samplefrom the culture or from the animal under control conditions (e.g., towhich no compound has been administered). Statistically significant(p<0.05) differences between the amount measured from the test sampleand from the control sample are recorded and indicate that the compoundalters the amount of Repro-EN-1.0 or IB1 produced by the cell.

The compound to be tested can be selected from a number of sources. Forexample, combinatorial libraries of molecules are available forscreening experiments. Using such libraries, thousands of molecules canbe screened for regulatory activity. In one preferred embodiment, highthroughput screening methods involve providing a library containing alarge number of potential therapeutic compounds (candidate compounds).Such “combinatorial chemical libraries” are then screened in one or moreassays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds” or can themselves be used as potential oractual therapeutics.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesisis by no means the only approach envisioned and intended for use withthe present invention. Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991),encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), randombio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries, peptide nucleic acid libraries (see, e.g., U.S. Pat. No.5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996) NatureBiotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al. (1996) Science, 274: 1520-1522, andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum (1993) C&EN, January 18, page 33,isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones and metathiazanonesU.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat. Nos. 5,525,735 and5,519,134, morpholino compounds U.S. Pat. No. 5,506,337, benzodiazepinesU.S. Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

In one embodiment this invention provides inhibitory compounds thatinhibit expression of Repro-EN-1.0 or IB1 identified or identifiable bythe screening methods of this invention.

XV. Genomics

The identification of cognate or polymorphic forms of the Repro-EN-1.0or IB1 gene and the tracking of those polymorphisms in individuals andfamilies is important in genetic screening. Accordingly, this inventionprovides methods useful in detecting polymorphic forms of theRepro-EN-1.0 or IB1 gene. The methods involve comparing the identity ofa nucleotide or amino acid at a selected position within the sequence ofa test Repro-EN-1.0 or IB1 gene with the nucleotide or amino acid at thecorresponding position from the sequence of native Repro-EN-1.0 (SEQ IDNO: 1) or IB1 (SEQ ID NO:3). The comparison can be carried out by anymethods known in the art, including direct sequence comparison bynucleotide sequencing, sequence comparison or determination byhybridization or identification of RFLPs.

In one embodiment, the method involves nucleotide or amino acidsequencing of the entire test polynucleotide or polypeptide, or asubsequence from it, and comparing that sequence with the sequence ofnative Repro-EN-1.0 or IB1. In another embodiment, the method involvesidentifying restriction fragments produced upon restriction enzymedigestion of the test polynucleotide and comparing those fragments withfragments produced by restriction enzyme digestion of nativeRepro-EN-1.0 or IB1 gene. Restriction fragments from the native gene canbe identified by analysis of the sequence to identify restriction sites.Another embodiment involves the use of oligonucleotide arrays. (See,e.g., Fodor et al., U.S. Pat. No. 5,445,934.) The method involvesproviding an oligonucleotide array comprising a set of oligonucleotideprobes that define sequences selected from the native Repro-EN-1.0 orIB1 sequence, generating hybridization data by performing ahybridization reaction between the target polynucleotide molecules andthe probes in the set and detecting hybridization between the targetmolecules and each of the probes in the set and processing thehybridization data to determine nucleotide positions at which theidentity of the target molecule differs from that of native Repro-EN-1.0or IB1. The comparison can be done manually, but is more convenientlydone by a programmable, digital computer.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLES I. Construction of a Human Endometrial Carcinoma Cell Line(RL95-2) cDNA Expression Library

A. Material and Methods

Poly A+ RNA isolated from RL95-2, was used as a template for firststrand cDNA synthesis. The poly A+ RNA was analyzed by denaturing gelelectrophoresis and ranged in size from 0.2 to 10 Kb (10). An oligo dTprimer containing an internal, protected Xhol site was annealed in thepresence of a nucleotide mixture containing 5-methyl dCTP, and extendedwith MMTV reverse transcriptase.

Second strand synthesis of the RL95-2 cDNA/RNA hybrid was completed bythe addition of RNase H, DNA polymerase 1, and dNTPs to the first strandsynthesis reaction. Pfu DNA polymerase was used to blunt-end the doublestranded RL95-2 cDNA followed by the ligation of EcoRl adapters. ThecDNA was kinased and digested with Xhol and EcoRl before sizefractionation on Sephacryl S-500 columns. The size fractionated cDNA wasrecovered and the quantified on ethidium bromide containing platesagainst a set of serially-diluted DNA standards. The cDNA contained inthe first two column fractionations was directionally ligated, in thesense orientation, to Xhol/EcoRl-digested uniZAP phage vector arms.Initially, approximately 25 ng (per fraction) of the cDNA was ligatedand packaged into bacteriophage particles. Subsequently, theapproximately 100 ng remaining cDNA in fractions 1 and 2 was packagedinto bacteriophage particles using several reactions of the lambda phagepackaging extract (Stratagene).

After packaging, the primary human RL95-2 library was titered byinfection of the XL1 Blue host strain. The ratio ofrecombinant:nonrecombinant phage was determined by plating infected XL1Blue in the presence of IPTG and XGal. The number of blue(non-recombinant) or white (recombinant) plaques were quantified using aManostat colony counter. Ninety-eight and one-half (98.5) percent of thephage in the human RL95-2 cDNA library were recombinant and the primaryphage library base consisted of 2.2×10⁶ independent clones. The humanRL95-2 cDNA library was amplified once and re-titered as above. Thetiter of the amplified library was 3×10⁹ pfu/ml.

The average size of the cDNA inserts was determined by PCRamplification. Twenty well-isolated phage plaques were cored and thecDNA inserts were amplified using T7/T3 specific oligonucleotides whichhybridize to sites flanking the cDNA insertion site contained within thelambda phage vector. The PCR products were analyzed by agarose gelelectrophoresis and visualized by ethidium bromide staining. Ninety-fivepercent of the cored plaques contained inserts varying in size from 0.5to 2.5 Kb with an average size of 1.5 Kb.

II. Library Screening

To identify endometrial autoantigens that could be used to develop anendometriosis immunoassay, pooled sera from patients (n=17) withlaparoscopy confirmed endometriosis was used as a probe to screen theRL95-2 cDNA expression library using the following methods.

A. Preadsorption of serum. Antibodies that react with expression libraryhost strains were immunoadsorbed from patient serum by using BNN97 andY1090 E. coli lysate-conjugated sepharose beads (5′→3′) following themanufacturer's protocols. Briefly, 2 ml of host stain lysate-conjugatedsepharose beads were washed twice with sterile Tris buffered saline(TBS). The beads were resuspended in 4 mls of serum diluted 1/2 in TBS.Following a 16 hour incubation at 4° C., the sepharose beads werecollected by centrifugation at 1,000×g for 2 min. The supernatant wasremoved and the beads were washed with 4 mls of sterile TBS. Aftercentrifugation at 1,000×g for 2 min, the supernatants were collected,pooled and used to screen the RL95-2-specific cDNA expression library.

B Screening the RL95-2 cDNA library. Approximately 10⁶ infectious phageparticles were incubated with XL-1 blue host cells and plated at densityof 50,000 phage per 150 mm dish using standard protocols (Stratagene).After incubating for 5 hours at 42° C., the phage plaques were overlaidwith nitrocellulose membranes (Protran; Schleicher & Schuell) that hadbeen soaked in a 10 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG)solution. Following a 4 hour incubation at 37° C., the membranes wereremoved and washed three times for 15 min in TBS with 0.05% Tween₂₀(TTBS). The membranes were incubated with blocking solution (1% Bovineserum albumin [fraction V] in TBS) for 1 hour at room temperature priorto a 1 hour incubation with preadsorbed patient serum diluted 1/10(final dilution of 1/40) in blocking solution. The membranes were washedthree time for 15 min with TTBS prior to the addition of alkalinephosphatase-conjugated goat anti-human Ig(G,A,M) (Pierce) diluted1/25,000 in blocking solution. After a 1 hour incubation at roomtemperature the membranes were washed three times with TTBS as describedabove and once with TBS. The membranes were incubated with enzymesubstrate (Western Blue; Promega) for approximately 30 min and theenzymatic reaction was terminated by briefly incubating the membraneswith stop solution (Tris-HCl pH 2.9; 1 mM EDTA). Several immunoreactivephage plaques were selected and transferred to 500 μl of SM buffer (100mM NaCl, 8 mM MgSO₄, 50 mM Tris-HCl pH 7.5, 0.01% gelatin) containing 20μl of chloroform. The selected phage were eluted from the agar andplated at a density of approximately 1,000 phage per 100 mm dish andscreen as described above. To insure that the selected phage plaque,named Repro-EN-1.0, represented a single clone the screening process wasrepeated a third time as described above.

C. Excision of Repro-EN-1.0 phagemid. Plasmid containing the cDNA insertfor Repro-EN-1.0 was excised from the phage clone using themanufacturer's protocols (Stratagene). The size of the Repro-EN-1.0insert was determined by releasing the cDNA fragment from the rescuedpBluescript/Repro-EN-1.0 plasmid with the restriction enzymes EcoRl andXhol. The released insert was size fractionated by agarose-gelelectrophoresis and the apparent length of the insert was determined bycomparing its migration position with a DNA standard (1 kb ladder; GibcoBRL). The insert migrated at approximately 2.0 Kb.

D. Identification of the IB1 clone. An alternately spliced variant ofRepro-EN-1.0. A commercial human heart cDNA library (Clontech) wasscreened with a radiolabeled probe mapping within the amino terminus ofthe Repro-EN-1.0 coding sequence (nt 203 to 897). One of the two clonesisolated contained a cDNA insert of 3.4 Kb which possessed an extra 231base pair insert within the Repro-EN-1.0 coding sequence.

III. Characterization

A. Sequence analysis

The nucleotide sequence of Repro-EN-1.0 was determined by using amodified protocol of the dideoxy chain termination method of Sanger etal. and USB Sequenase 2.0 (Barker, D. F. 1993, Biotechniques). The aminoacid sequence was predicted using the Intelligenetic TRANSLATE programand sequence homologies were determined with BLAST data base searchalgorithms. The deduced amino acid sequence (in the expected frame for afusion protein) was novel.

B. Tissue Expression Analysis

The Repro-EN-1.0 expression distribution was determined by Northern blotanalysis using poly A+ RNA collected from human spleen, thymus,prostate, testis, ovary, small intestine, colon, and peripheral bloodleukocytes (MTN human blot 11; Clontech) and human heart, brain,placenta, lung, liver, skeletal muscle, kidney and pancreas (MTN humanblot 1-Clontech) and the manufacturer' suggested protocols (Clontech).The immobilized poly A+ RNA samples were incubated with a random primelabeled probe that represented the Repro-EN-1.0 insert. The probe wasgenerated by using and EcoRl/Xhol released fragment of pRepro-EN-1.0 astemplate for a random prime labeled reaction as described in themanufacturer's manual (Megaprime kit; Amersham). The Northern blotmembranes were prehybridized with 6 mls of ExpressHyb solution(Clontech) followed by a 1 hour incubation at 68° C. with approximately0.5 ng of radiolabeled probe (approx. 2×105 cpm) in 5 mls of ExpressHybsolution. Unbound probe was removed by washing the membranes three timeswith 2×SSC, 0.05% SDS at room temperature for 10 min for each washfollowed by twice with 0.1×SSC, 0.1% SDS at 50° C. for 15 min for eachwash. The apparent length of RNA species, a 3.4 Kb mRNA, and tissuedistribution was determined by autoradiography. Expression was detectedprimarily in skeletal muscle, heart and testis; and to a lesser extentin other tissues, but was not detected in lung or peripheral bloodmononuclear cells (PBMC). Expression of Repro-EN-1.0 was up-regulated inbreast and uterine carcinomas relative to their normal counterparts, washighly expressed in both normal fallopian tube and fallopian tubecarcinoma, and was expressed at low levels in both normal ovary andovarian carcinoma (FIG. 2 and FIG. 3). Expression of Repro-EN-1.0 inRL95-2 (endometrium carcinoma) cells is lower than in LNCaP (humanprostate adenocarcinoma), PC-12 (rat cell line) and BT12 (human breastcarcinoma cell line) cells and undetectable in a mouse hybridoma cellline (3E10; negative control) (FIG. 4). In addition, expression innormal endometrium is undetectable.

C. Homologue Analysis

To determine the level of nucleotide conservation of Repro-EN-1.0 indifferent species, a Southern blot analysis using EcoRl digested genomicDNA collected from human, monkey, rat, mouse, dog, cow, rabbit, chickenand yeast was performed as described in the manufacture's manual(ZooBlot; Clontech). Briefly, the immobilized EcoRl digested genomic DNAsamples were prehybridized with 6 mls of ExpressHyb (Clontech) andincubated for 1 hour at 68° C. with 1.0 ng (approx. 4×10⁵ cpm in 5 mls)of a random prime labeled probe that represented the Repro-EN-1.0insert. Unbound probe was removed by washing the membranes once with2×SSC, 0.05% SDS at room temperature for 30 min followed by one washwith 0.1×SSC, 0.1% SDS at 50 C for 30 min. Identification of homologuesin different species was determine by autoradiography. (See FIG. 4) Thesequence is highly conserved between human and non-human primates(Monkey).

IV. Antibodies

Antibodies to peptides of the clone and/or to recombinant protein weregenerated in rabbits. This antisera was used to develop a Repro-EN-1.0ELISA.

V. Recombinant Protein

The predicted ORF for Repro-EN-1 was subcloned into an expressionvector, and the recombinant protein was expressed and purified byNi-Chelate chromatography using standard methodologies.

VI. ELISA

The purified recombinant Repro-EN-1 protein was used as a target antigenin an ELISA (EndX™ ELISA) designed to detect antigen-specificautoantibodies in patient serum.

The present invention provides a novel nucleotide sequence encodingRepro-EN-1.0, Repro-EN-1.0 polypeptides, IB1, IB1 polypeptides andmethods of using these materials. While specific examples have beenprovided, the above description is illustrative and not restrictive.Many variations of the invention will become apparent to those skilledin the art upon review of this specification. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument Applicants do not admit that any particular reference is “priorart” to their invention.

1. A method for diagnosing endometriosis in a human subject comprisingthe steps of: (a) detecting a test amount of an antibody thatspecifically binds to SEQ ID NO: 2 or SEQ ID NO: 4 polypeptide or atruncated peptide comprising an epitope of SEQ ID NO: 2 or SEQ ID NO: 4polypeptide, in a sample from the subject; and (b) comparing the testamount with a normal range of the antibody in a control sample from asubject who does not suffer from endometriosis, whereby a test amountabove the normal range of the antibody provides a positive indication inthe diagnosis of endometriosis.
 2. The method of claim 1, wherein thesample comprises blood serum, peritoneal fluid, menstrual fluid, vaginalsecretion or urine.
 3. The method of claim 1 or 2, wherein the step ofdetecting comprises capturing the antibody from the sample with saidpolypeptide or peptide which has been immobilized and detecting capturedantibody.
 4. The method of claim 1 wherein said polypeptide has theamino acid sequence identical to SEQ ID NO:2.
 5. The method of claim 1wherein said polypeptide has the amino acid sequence identical to SEQ IDNO:
 4. 6. The method of claim 1 wherein said truncated peptide comprisesan epitope of SEQ ID NO:
 2. 7. The method of claim 1 wherein saidtruncated peptide comprises an epitope of SEQ ID NO:
 4. 8. The method ofclaim 1 wherein said SEQ ID NO: 2 or SEQ ID NO: 4 polypeptide isproduced by recombinant means.
 9. The method of claim 1 wherein saidtruncated peptide of SEQ ID NO: 2 or SEQ ID NO: 4 is produced byrecombinant means.